Bio

Academic Appointments


Administrative Appointments


  • Chair, Department of Biological Sciences, Stanford University (1982 - 1989)
  • Chair, Second Senate ad hoc Committee on the Professoriate, Stanford University (1988 - 1990)
  • Director, Biophysics Graduate Program, Stanford University (1968 - 1985)
  • Member, School Planning Group, Humanities and Sciences, Stanford Universtiy (1991 - 1993)
  • Board of Trustees, Oberlin College (1998 - 2007)
  • Editorial Board, Proceedings of the National Academy of Sciences USA (2000 - Present)
  • Senior Editor, Cancer Research, American Association for Cancer Research (2003 - 2010)
  • Pre-doctoral Fellowship Review Panel, National Science Foundation (1985 - 1986)
  • Chair, External Advisory Board, Program on Structural Biology of DNA Repair, Lawrence Berkeley National Laboratory (2001 - Present)
  • Chair, US National Committee, International Union of Pure and Applied Biophysics (1969 - 1975)
  • Chemical Pathology Study Section, National Institutes of Health (1981 - 1984)
  • Physiological Chemistry Study Section, National Institutes of Health (1966 - 1970)
  • Council for Extramural Grants, American Cancer Society (1998 - 2001)
  • Member, 23rd Senate of the Academic Council, Stanford University (1990 - 1992)
  • Chair, Administrative Panel on Radiological Hazards, Stanford University (1978 - 1980)
  • Board of Directors, American Association for Cancer Research (1994 - 1997)
  • International Advisory Board, Chulabhorn Research Institute (2005 - 2010)
  • Consultant, Achaogen (2005 - 2006)
  • External Advisory Comm, MD Anderson Cancer Center (2004 - 2007)
  • Abbott-ASM Lifetime Ach Selection Committee, American Academy of Microbiology (2003 - 2006)
  • Council for Extramural Grants, Amercian Cancer Society (1998 - 2001)
  • NRC Committee, BEIR VII Phase I (1997 - 1998)
  • Scientific Advisory Board, Forgarty International Center, NIH (1995 - 1999)
  • Toxicology Advisory Board, The Burroughs Wellcome Fund (1995 - 2004)
  • External Advisory comm, City of Hope Cancer Center (1995 - 2007)
  • Carcinogen Carcinogen Identification Committee and AdvisoryBoard, CA-EPA (1994 - 1998)
  • Chair, External Advisory Brd, University of Texas Medical Branch (1994 - 1997)

Honors & Awards


  • AACR-Princess Takamatsu Lectureship, American Association for Cancer Research (April 2011)
  • The Dr. Morris Herzstein Professorship in Biology, Stanford University (2008 -)
  • Howard H. and Jessie T. Watkins University Professor, Stanford University (1997 - 2002)
  • Member, National Academy of Sciences, USA (1989)
  • Peter and Helen Bing Award for Distinguished Teaching, Stanford University (1992)
  • Excellence in Teaching Award, Northern California Chapter, Phi Beta Kappa (1991)
  • Outstanding Investigator Research Award, National Cancer Institute, NIH (1987 - 2001)
  • International Mutation Research Achievement Award, Elsevier (1997)
  • Senior Scholar Research Award, Ellison Medical Foundation (2001- 2005)
  • Annual Award for Excellence in Basic Science, Environmental Mutagen Society (1992)
  • Keynote Lecture, 10th International Conference on Environmental Mutagens, Florence, Italy (2009)
  • Keynote Lecture, ASM International Conference on DNA repair and mutagenesis, Bermuda (2004)
  • Chair, Gordon Conference on Mutagenesis (1996)
  • Chair, Gordon Conference on Mammalian DNA Repair (1999)
  • Rothschild-Yvette Mayent- Institute Curie Award/Lectureship, Curie Institute. Paris, France (2003)
  • President, Environmental Mutagen Society (1994)
  • Annual Research Award, American Society for Photobiology (1996)
  • John B Little Award in Radiation Health Sciences, Harvard School of Public Health (2002)
  • Doctor Honoris Causa, University of Bio Bio, Concepcion, Chile (2006)
  • Honorary Doctor of Science, Oberlin College (1997)
  • Fellow, American Academy of Microbiology (1993)
  • Foreign Associate, European Molecular Biology Organization (2001)
  • Doctor Honoris Causa, University of Sevile, Sevile, Spain (2008)
  • Fellow, American Acaemy of Arts and Sciences (2008)
  • Lectureship, Spanish Academy of Science & Catalan Society (1982)
  • Inaugural Annual Lecture, Lord Dowding Fund for Humane Research (1982)
  • Fellow, American Association for Advancement of Sciences (1981)

Professional Education


  • Ph.D., Yale University, Biophysics (1959)
  • M.S., Yale University, Physics (1955)
  • B.A., Oberlin College, Physics (1954)

Research & Scholarship

Current Research and Scholarly Interests


Hanawalt has been a productive researcher in the field of DNA repair since his pioneering discovery of repair replication in E. coli in 1963. In 1982 Hanawalt and his colleagues reported the first example of intragenomic DNA repair heterogeneity: chemical adducts in alpha DNA in African green monkey kidney cells were not as efficiently repaired as in the genome overall. Hanawalt and his colleagues then discovered that repair of some types of damage is selective; active genes are preferentially repaired, and in fact a special repair pathway, termed transcription-coupled repair (TCR), operates on the transcribed strands of expressed genes. TCR was documented in mammalian cells, in E. coli, and in yeast chromosomal and plasmid borne genes. The discovery of TCR in Hanawalt’s laboratory has had profound implications for the fields of mutagenesis, environmental carcinogenesis, aging, and risk assessment.
The prototype recQ gene was discovered in E. coli in Hanawalt’s laboratory, and we now know of five homologues in humans including the genes mutated in the cancer prone hereditary diseases: Bloom’s syndrome, Werner’s syndrome, and Rothman Thompson syndrome.
More recent studies have focused upon the regulation of TCR and the global genomic nucleotide excision repair (GGR) pathway. Features of the TCR pathway (defective in Cockayne syndrome) include the possibility of "gratuitous TCR" at transcription pause sites in undamaged DNA. The GGR pathway was shown to be controlled through the SOS stress response in E. coli and through the activated product of the p53 tumor suppressor gene in human cells. These regulatory systems particularly affect the efficiency of repair of the predominant UV-induced photoproduct, the cyclobutane pyrimidine dimer, as well as that of chemical carcinogen DNA adducts, such as benzo(a)pyrene diol-epoxide and benzo(g)chrysene. Rodent cells (typically lacking the p53-controlled GGR pathway) are unable to carry out efficient GGR of some lesions. Therefore, caution should be exercised in the interpretation of results from such systems for risk assessment in genetic toxicology.

Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • DNA slip-outs cause RNA polymerase II arrest in vitro: potential implications for genetic instability NUCLEIC ACIDS RESEARCH Salinas-Rios, V., Belotserkovskii, B. P., Hanawalt, P. C. 2011; 39 (17): 7444-7454

    Abstract

    The abnormal number of repeats found in triplet repeat diseases arises from 'repeat instability', in which the repetitive section of DNA is subject to a change in copy number. Recent studies implicate transcription in a mechanism for repeat instability proposed to involve RNA polymerase II (RNAPII) arrest caused by a CTG slip-out, triggering transcription-coupled repair (TCR), futile cycles of which may lead to repeat expansion or contraction. In the present study, we use defined DNA constructs to directly test whether the structures formed by CAG and CTG repeat slip-outs can cause transcription arrest in vitro. We found that a slip-out of (CAG)(20) or (CTG)(20) repeats on either strand causes RNAPII arrest in HeLa cell nuclear extracts. Perfect hairpins and loops on either strand also cause RNAPII arrest. These findings are consistent with a transcription-induced repeat instability model in which transcription arrest in mammalian cells may initiate a 'gratuitous' TCR event leading to a change in repeat copy number. An understanding of the underlying mechanism of repeat instability could lead to intervention to slow down expansion and delay the onset of many neurodegenerative diseases in which triplet repeat expansion is implicated.

    View details for DOI 10.1093/nar/gkr429

    View details for Web of Science ID 000295184800015

    View details for PubMedID 21666257

  • Anchoring Nascent RNA to the DNA Template Could Interfere with Transcription BIOPHYSICAL JOURNAL Belotserkovskii, B. P., Hanawalt, P. C. 2011; 100 (3): 675-684

    Abstract

    During normal transcription, the nascent RNA product is released from the DNA template. However, in some cases, the RNA remains bound or can become reattached to the template DNA duplex (for example, through R-loop formation). We have analyzed the effect on transcription elongation of nascent RNA anchoring to the template DNA duplex. Because the RNA polymerase follows a helical path along DNA duplex during transcription, the anchoring would result in wrapping the nascent RNA around the DNA in the region between the anchoring point and the translocating polymerase. This wrapping would cause an unfavorable loss of conformation entropy of the nascent RNA. It consequently would create an apparent force to unwrap the RNA by disrupting either the transcription complex or the anchoring structure. We have estimated that this force would be comparable to those required to melt nucleic acid duplexes or to arrest transcription elongation in single-molecule experiments. We predict that this force would create negative supercoiling in the DNA duplex region between the anchoring point and the transcribing RNA polymerase: this can promote the formation of unusual DNA structures and facilitate RNA invasion into the DNA duplex. Potential biological consequences of these effects are discussed.

    View details for DOI 10.1016/j.bpj.2010.12.3709

    View details for Web of Science ID 000286957200018

    View details for PubMedID 21281582

  • Transcription-coupled nucleotide excision repair of a gene transcribed by bacteriophage T7 RNA polymerase in Escherichia coli DNA REPAIR Ganesan, A. K., Hanawalt, P. C. 2010; 9 (9): 958-963

    Abstract

    Transcription-coupled nucleotide excision repair (TC-NER) removes certain kinds of lesions from the transcribed strand of expressed genes. The signal for TC-NER is thought to be RNA polymerase stalled at a lesion in the DNA template. In Escherichia coli, the stalled polymerase is dissociated from the lesion by the transcription repair coupling factor (Mfd protein), which also recruits excision repair proteins to the site resulting in efficient removal of the lesion. TC-NER has been documented in cells from a variety of organisms ranging from bacteria to humans. In each case, the RNA polymerase involved has been a multimeric protein complex. To ascertain whether a gene transcribed by the monomeric RNA polymerase of bacteriophage T7 could be repaired by TC-NER, we constructed strains of E. coli in which the chromosomal lacZ gene is controlled by a T7 promoter. In the absence of T7 RNA polymerase, little or no beta-galactosidase is produced, indicating that the E. coli RNA polymerase does not transcribe lacZ efficiently, if at all, in these strains. By introducing a plasmid (pAR1219) carrying the T7 gene 1 under control of the E. coli lac UV5 promoter into these strains, we obtained derivatives in which the level of T7 RNA polymerase could be regulated. In cultures containing upregulated levels of the polymerase, beta-galactosidase was actively produced indicating that the T7 RNA polymerase transcribes the lacZ gene efficiently. Under these conditions, we observed that UV-induced cyclobutane pyrimidine dimers were removed more rapidly from the transcribed strand of lacZ than from the nontranscribed strand, supporting the conclusion that TC-NER occurred in this gene. This response was absent in an mfd-1 mutant, indicating that the underlying mechanism may be similar to that for the bacterial RNA polymerase.

    View details for DOI 10.1016/j.dnarep.2010.06.007

    View details for Web of Science ID 000282078100003

    View details for PubMedID 20638914

  • Mechanisms and implications of transcription blockage by guanine-rich DNA sequences PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Belotserkovskii, B. P., Liu, R., Tornaletti, S., Krasilnikova, M. M., Mirkin, S. M., Hanawalt, P. C. 2010; 107 (29): 12816-12821

    Abstract

    Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and with genomic instability. An important example is sequences containing G-rich homopurine-homopyrimidine stretches, for which unusual transcriptional behavior is implicated in regulation of immunogenesis and in other processes such as genomic translocations and telomere function. To elucidate the mechanism of the effect of these sequences on transcription we have studied T7 RNA polymerase transcription of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, length- and supercoiling-dependent manner. Based upon the effects of various sequence modifications, solution conditions, and ribonucleotide substitutions, we conclude that transcription blockage is due to formation of unusually stable RNA/DNA hybrids, which could be further exacerbated by triplex formation. These structures are likely responsible for transcription-dependent replication blockage by G-rich sequences in vivo.

    View details for DOI 10.1073/pnas.1007580107

    View details for Web of Science ID 000280144500022

    View details for PubMedID 20616059

  • Peptide Nucleic Acid (PNA) Binding and Its Effect on In Vitro Transcription in Friedreich's Ataxia Triplet Repeats MOLECULAR CARCINOGENESIS Belotserkovskii, B. P., Liu, R., Hanawalt, P. C. 2009; 48 (4): 299-308

    Abstract

    Peptide nucleic acids (PNAs) are DNA mimics in which peptide-like linkages are substituted for the phosphodiester backbone. Homopyrimidine PNAs can invade double-stranded DNA containing the homologous sequence by displacing the homopyrimidine strand from the DNA duplex and forming a PNA/DNA/PNA triplex with the complementary homopurine strand. Among biologically interesting targets for triplex-forming PNA are (GAA/CTT)(n) repeats. Expansion of these repeats results in partial inhibition of transcription in the frataxin gene, causing Friedreich's ataxia. We have studied PNA binding and its effect on T7 RNA polymerase transcription in vitro for short repeats (n = 3) and for long repeats (n = 39), placed in both possible orientations relative to the T7 promoter such that either the GAA-strand, or the CTT-strand serves as the template for transcription. In all cases PNA bound specifically and efficiently to its target sequence. For the short insert, PNA binding to the template strand caused partial transcription blockage with well-defined sites of RNA product truncation in the region of the PNA-binding sequence, whereas binding to the nontemplate strand did not block transcription. However, PNA binding to long repeats, whether in the template or the nontemplate strand, resulted in a dramatic reduction of the amount of full-length transcription product, although in the case of the nontemplate strand there were no predominant truncation sites. Biological implications of these results are discussed.

    View details for DOI 10.1002/mc.20486

    View details for Web of Science ID 000264918500004

    View details for PubMedID 19306309

  • New applications of the Comet assay: Comet-FISH and transcription-coupled DNA repair MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH Spivak, G., Cox, R. A., Hanawalt, P. C. 2009; 681 (1): 44-50

    Abstract

    Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30kb. The single cell gel electrophoresis (Comet) assay allows detection of single- or double-strand breaks at a 10-100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet "heads", and that Comet "tails" consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR.

    View details for DOI 10.1016/j.mrrev.2007.12.003

    View details for Web of Science ID 000263813200006

    View details for PubMedID 18291710

  • Transcription-coupled DNA repair: two decades of progress and surprises NATURE REVIEWS MOLECULAR CELL BIOLOGY Hanawalt, P. C., Spivak, G. 2008; 9 (12): 958-970

    Abstract

    Expressed genes are scanned by translocating RNA polymerases, which sensitively detect DNA damage and initiate transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes lesions from the template DNA strands of actively transcribed genes. Human hereditary diseases that present a deficiency only in TCR are characterized by sunlight sensitivity without enhanced skin cancer. Although multiple gene products are implicated in TCR, we still lack an understanding of the precise signals that can trigger this pathway. Futile cycles of TCR at naturally occurring non-canonical DNA structures might contribute to genomic instability and genetic disease.

    View details for DOI 10.1038/nrm2549

    View details for Web of Science ID 000261126800015

    View details for PubMedID 19023283

  • Emerging links between premature ageing and defective DNA repair MECHANISMS OF AGEING AND DEVELOPMENT Hanawalt, P. C. 2008; 129 (7-8): 503-505

    View details for DOI 10.1016/j.mad.2008.03.007

    View details for Web of Science ID 000257816100017

    View details for PubMedID 18440595

  • Inhibitory effect of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase NUCLEIC ACIDS RESEARCH Ditlevson, J. V., Tornaletti, S., Belotserkovskii, B. P., Teijeiro, V., Wang, G., Vasquez, K. M., Hanawalt, P. C. 2008; 36 (10): 3163-3170

    Abstract

    DNA sequences capable of forming unusual secondary structures can be a source of genomic instability. In some cases that instability might be affected by transcription, as recently shown for the Z-DNA forming sequence (CG)(14), which causes genomic instability both in mammalian cells and in bacteria, and this effect increases with its transcription. We have investigated the effect of this (CG)(14) sequence on transcription with T7 RNA polymerase in vitro. We detected partial transcription blockage within the sequence; the blockage increased with negative supercoiling of the template DNA. This effect was not observed in a control self-complementary sequence of identical length and base composition as the (CG)(14) sequence, when the purine-pyrimidine alternation required for Z-DNA formation was disrupted. These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)(14) sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product.

    View details for DOI 10.1093/nar/gkn136

    View details for Web of Science ID 000257183200001

    View details for PubMedID 18400779

  • G4-forming sequences in the non-transcribed DNA strand pose blocks to T7 RNA polymerase and mammalian RNA polymerase II JOURNAL OF BIOLOGICAL CHEMISTRY Tornaletti, S., Park-Snyder, S., Hanawalt, P. C. 2008; 283 (19): 12756-12762

    Abstract

    DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

    View details for DOI 10.1074/jbc.M705003200

    View details for Web of Science ID 000255499800011

    View details for PubMedID 18292094

  • Paradigms for the three Rs: DNA replication, recombination, and repair MOLECULAR CELL Hanawalt, P. C. 2007; 28 (5): 702-707

    Abstract

    The recent decade has engendered a convergence of the otherwise distinct fields of DNA replication, recombination, and repair, as we are learning how these essential transactions can operate in coordination to achieve genomic stability and to ensure cellular viability. In the next decade, we can anticipate a functional understanding of the roles of posttranslational protein modifications in the regulation and prioritizing of pathways for genomic maintenance. The fundamental knowledge gained should lead to more effective clinical intervention in human disease.

    View details for DOI 10.1016/j.molcel.2007.11.014

    View details for Web of Science ID 000251926000002

    View details for PubMedID 18082594

  • A triplex-forming sequence from the human c-MYC promoter interferes with DNA transcription JOURNAL OF BIOLOGICAL CHEMISTRY Belotserkovskii, B. P., De Silva, E., Tornaletti, S., Wang, G., Vasquez, K. M., Hanawalt, P. C. 2007; 282 (44): 32433-32441

    Abstract

    Naturally occurring DNA sequences that are able to form unusual DNA structures have been shown to be mutagenic, and in some cases the mutagenesis induced by these sequences is enhanced by their transcription. It is possible that transcription-coupled DNA repair induced at sites of transcription arrest might be involved in this mutagenesis. Thus, it is of interest to determine whether there are correlations between the mutagenic effects of such noncanonical DNA structures and their ability to arrest transcription. We have studied T7 RNA polymerase transcription through the sequence from the nuclease-sensitive element of the human c-MYC promoter, which is mutagenic in mammalian cells (Wang, G., and Vasquez, K. M. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13448-13453). This element has two mirror-symmetric homopurine-homopyrimidine blocks that potentially can form either DNA triplex (H-DNA) or quadruplex structures. We detected truncated transcription products indicating partial transcription arrest within and closely downstream of the element. The arrest required negative supercoiling and was much more pronounced when the pyrimidine-rich strand of the element served as the template. The exact positions of arrest sites downstream from the element depended upon the downstream flanking sequences. We made various nucleotide substitutions in the wild-type sequence from the c-MYC nuclease-sensitive element that specifically destabilize either the triplex or the quadruplex structure. When these substitutions were ranked for their effects on transcription, the results implicated the triplex structure in the transcription arrest. We suggest that transcription-induced triplex formation enhances pre-existing weak transcription pause sites within the flanking sequences by creating steric obstacles for the transcription machinery.

    View details for DOI 10.1074/jbc.M704618200

    View details for Web of Science ID 000250480300066

    View details for PubMedID 17785457

  • Transcription coupled nucleotide excision repair in Escherichia coli can be affected by changing the arginine at position 529 of the p subunit of RNA polymerase DNA REPAIR Ganesan, A. K., Smith, A. J., Savery, N. J., Zamos, P., Hanawalt, P. C. 2007; 6 (10): 1434-1440

    Abstract

    The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the beta subunit of RNAP; changing any one of these to alanine diminishes the interaction [1]. Using direct assays for TCR, and the lac operon of E. coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the beta subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR.

    View details for DOI 10.1016/j.dnarep.2007.04.002

    View details for Web of Science ID 000250080300005

    View details for PubMedID 17532270

  • Nucleotide excision repair phenotype of human acute myeloid leukemia cell lines at various stages of differentiation MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS Hsu, P., Hanawalt, P. C., Nouspikel, T. 2007; 614 (1-2): 3-15

    Abstract

    In previous studies it was shown that nucleotide excision repair (NER) is strongly attenuated at the global genome level in terminally differentiated neuron-like cells. NER was measured in several human acute myeloid leukemia cell lines, before and after differentiation into macrophage-like cells. Repair of cisplatin intrastrand GTG crosslinks in differentiated cells was strongly attenuated. There were also some variations between repair levels in naïve cells, but these were not correlated with the degree of differentiation. By contrast, the proficient repair of UV-induced (6-4)pyrimidine-pyrimidone photoproducts [(6-4)PPs] was not affected by differentiation. Although cyclobutane pyrimidine dimers (CPDs) were poorly repaired at the global genome level in all cell lines, differentiated or not, they were very efficiently removed from the transcribed strand of an active gene, indicating that transcription-coupled repair (TCR) is proficient in each cell line. CPDs were also removed from the non-transcribed strand of an active gene better than at the overall global genome level. This relatively efficient repair of the non-transcribed strand of active genes, when compared with global genomic repair (GGR), has been described previously in neuron-like cells and termed differentiation-associated repair (DAR). Here we show that it also can occur in actively growing cells that display poor GGR.

    View details for DOI 10.1016/j.mrfmmm.2006.06.008

    View details for Web of Science ID 000243544900002

    View details for PubMedID 16890248

  • Transcription domain-associated repair in human cells MOLECULAR AND CELLULAR BIOLOGY Nouspikel, T. P., Hyka-Nouspikel, N., Hanawalt, P. C. 2006; 26 (23): 8722-8730

    Abstract

    Nucleotide excision repair (NER), which is arguably the most versatile DNA repair system, is strongly attenuated in human cells of the monocytic lineage when they differentiate into macrophages. Within active genes, however, both DNA strands continue to be proficiently repaired. The proficient repair of the nontranscribed strand cannot be explained by the dedicated subpathway of transcription-coupled repair (TCR), which is targeted to the transcribed strand in expressed genes. We now report that the previously termed differentiation-associated repair (DAR) depends upon transcription, but not simply upon RNA polymerase II (RNAPII) encountering a lesion: proficient repair of both DNA strands can occur in a part of a gene that the polymerase never reaches, and even if the translocation of RNAPII is blocked with transcription inhibitors. This suggests that DAR may be a subset of global NER, restricted to the subnuclear compartments or chromatin domains within which transcription occurs. Downregulation of selected NER genes with small interfering RNA has confirmed that DAR relies upon the same genes as global genome repair, rather than upon TCR-specific genes. Our findings support the general view that the genomic domains within which transcription is active are more accessible than the bulk of the genome to the recognition and repair of lesions through the global pathway and that TCR is superimposed upon that pathway of NER.

    View details for DOI 10.1128/MCB.01263-06

    View details for Web of Science ID 000242203700005

    View details for PubMedID 17015469

  • Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nouspikel, T., Hanawalt, P. C. 2006; 103 (44): 16188-16193

    Abstract

    Global nucleotide excision repair is greatly attenuated in terminally differentiated mammalian cells. We observed this phenomenon in human neurons and in macrophages, noting that the transcription-coupled repair pathway remains functional and that there is no significant reduction in levels of excision repair enzymes. We have discovered that ubiquitin-activating enzyme E1 complements the repair deficiency in macrophage extracts, and although there is no reduction in the concentration of E1 upon differentiation, our results indicate a reduction in phosphorylation of E1. In preliminary studies, we have identified the basal transcription factor TFIIH as the potential target for ubiquitination. We suggest that this unusual type of regulation at the level of the E1 enzyme is likely to affect numerous cellular processes and may represent a strategy to coordinate multiple phenotypic changes upon differentiation by using E1 as a "master switch."

    View details for DOI 10.1073/pnas.0607769103

    View details for Web of Science ID 000241879500025

    View details for PubMedID 17060614

  • Transcription arrest at an abasic site in the transcribed strand of template DNA CHEMICAL RESEARCH IN TOXICOLOGY Tornaletti, S., Maeda, L. S., Hanawalt, P. C. 2006; 19 (9): 1215-1220

    Abstract

    A dedicated excision repair pathway, termed transcription-coupled repair (TCR), targets the removal of DNA lesions from transcribed strands of expressed genes. Transcription arrest at the site of the lesion has been proposed as the first step for initiation of TCR. In support of this model, a strong correlation between arrest of transcription by a lesion in vitro and TCR of that lesion in vivo has been found in most cases analyzed. TCR has been reported for oxidative DNA damage; however, very little is known about how frequently occurring and spontaneous DNA damage, such as depurination and base deamination, affects progression of the transcription complex. We have previously determined that the oxidative lesion, thymine glycol, is a significant block to transcription by T7 RNA polymerase (T7 RNAP) but has no detectable effect on transcription by RNA polymerase II (RNAP II) in a reconstituted system with all of the required factors. Another oxidative lesion, 8-oxoguanine, only slightly blocked T7 RNAP and caused RNAP II to briefly pause at the lesion before bypassing it. Because an abasic site is an intermediate in the repair of oxidative damage, it was of interest to learn whether it arrested transcription. Using in vitro transcription assays and substrates containing a specifically positioned lesion, we found that an abasic site in the transcribed strand is a 60% block to transcription by T7 RNAP but nearly a complete block to transcription by mammalian RNAP II. An abasic site in the nontranscribed strand did not block either polymerase. Our results clearly indicate that an abasic site is a much stronger block to transcription than either a thymine glycol or an 8-oxoguanine. Because the predominant model for TCR postulates that only lesions that block RNAP will be subject to TCR, our findings suggest that the abasic site may be sufficient to initiate TCR in vivo.

    View details for DOI 10.1021/tx060103g

    View details for Web of Science ID 000240543400014

    View details for PubMedID 16978026

  • Research collaborations: Trial, trust, and truth CELL Hanawalt, P. C. 2006; 126 (5): 823-825

    Abstract

    Successful advances in biomedical research increasingly require multigroup collaborations and publication of results in multiauthored papers. It is essential to consider at the outset how to maximize the value of such collaborations while avoiding potential pitfalls.

    View details for DOI 10.1016/j.cell.2006.08.018

    View details for Web of Science ID 000240675000005

    View details for PubMedID 16959557

  • Role of DNA replication and repair in thymineless death in Escherichia coli JOURNAL OF BACTERIOLOGY Morganroth, P. A., Hanawalt, P. C. 2006; 188 (14): 5286-5288

    Abstract

    Inhibition of DNA replication with hydroxyurea during thymine starvation of Escherichia coli shows that active DNA synthesis is not required for thymineless death (TLD). Hydroxyurea experiments and thymine starvation of lexA3 and uvrA DNA repair mutants rule out unbalanced growth, the SOS response, and nucleotide excision repair as explanations for TLD.

    View details for DOI 10.1128/JB.00543-06

    View details for Web of Science ID 000239079300029

    View details for PubMedID 16816201

  • Topoisomerase deficiencies subtly enhance global genomic repair of ultraviolet-induced DNA damage in Saccharomyces cerevisiae DNA REPAIR Cline, S. D., Hanawalt, P. C. 2006; 5 (5): 611-617

    Abstract

    Genetic integrity depends upon the precision of all pathways that manipulate DNA. DNA repair mechanisms prevent mutations and aberrant recombination events by removing DNA damage. DNA topoisomerases maintain favorable nucleic acid topology for replication, transcription, and chromosome segregation. However, topoisomerases can also become trapped on DNA at sites of damage, and thereby, might alter the efficiency of DNA repair. The activities of the three nuclear DNA topoisomerases (Top1, Top2, and Top3) in the yeast Saccharomyces cerevisiae were examined for their influence upon the nucleotide excision repair (NER) of DNA damage induced by ultraviolet (UV) irradiation. A 10-20% increase in the global genomic repair (GGR) of cyclobutane pyrimidine dimers (CPDs) was observed with impaired Top1 or Top2 function. The GGR of 6-4 photoproducts (6-4PPs) and the strand-specific removal of CPDs from the yeast RPB2 gene were unaffected by the loss of topoisomerase activity. Even though the deletion of TOP3 conferred UV sensitivity, neither the GGR nor the strand-specific repair of UV-induced DNA damage was compromised in top3Delta yeast. Top1 and Top2 in DNA complexes near CPDs may inhibit GGR recognition of these lesions and produce protein-linked DNA breaks, resulting in CPD repair by an alternate pathway. While the physiological role of topoisomerase association with DNA damage has yet to be determined, these enzymes do not play a direct role in the NER pathways for removing UV-induced lesions in yeast.

    View details for DOI 10.1016/j.dnarep.2006.01.007

    View details for Web of Science ID 000237683700009

    View details for PubMedID 16516562

  • Transcriptional inhibition by an oxidized abasic site in DNA CHEMICAL RESEARCH IN TOXICOLOGY Wang, Y. L., Sheppard, T. L., Tornaletti, S., Maeda, L. S., Hanawalt, P. C. 2006; 19 (2): 234-241

    Abstract

    2-Deoxyribonolactone (dL) is an oxidized abasic site in DNA that can be induced by gamma-radiolysis, ultraviolet irradiation, and numerous antitumor drugs. Although this lesion is incised by AP endonucleases, suggesting a base-excision repair mechanism for dL removal, subsequent excision and repair synthesis by DNA polymerase beta is inhibited due to accumulation of a protein-DNA cross-link. This raises the possibility that additional repair pathways might be required to eliminate dL from the genome. Transcription-coupled repair (TCR) is a pathway of excision repair specific to DNA lesions present in transcribed strands of expressed genes. A current model proposes that transcription arrest at the site of DNA damage is required to initiate TCR. In support of this model, a strong correlation between transcription arrest by a lesion in vitro and TCR of the lesion in vivo has been found in most cases analyzed. To assess whether dL might be subject to TCR, we have studied the behavior of bacteriophage T3 and T7 RNA polymerases (T3RNAP, T7RNAP) and of mammalian RNA polymerase II (RNAPII) when they encounter a dL lesion or its "caged" precursor located either in the transcribed or in the nontranscribed strand of template DNA. DNA plasmids containing a specifically located dL downstream of the T3, T7 promoter or the Adenovirus major late promoter were constructed and used for in vitro transcription with purified proteins. We found that both dL and its caged precursor located in the transcribed strand represented a complete block to transcription by T3- and T7RNAP. Similarly, they caused more than 90% arrest when transcription was carried out with mammalian RNAPII. Furthermore, RNAPII complexes arrested at dL were subject to the transcript cleavage reaction mediated by elongation factor TFIIS, indicating that these complexes were stable. A dL in the nontranscribed strand did not block either polymerase.

    View details for DOI 10.1021/tx050292n

    View details for Web of Science ID 000235584800006

    View details for PubMedID 16485899

  • Host cell reactivation of plasmids containing oxidative DNA lesions is defective in Cockayne syndrome but normal in UV-sensitive syndrome fibroblasts DNA REPAIR Spivak, G., Hanawalt, P. C. 2006; 5 (1): 13-22

    Abstract

    UV-sensitive syndrome (UV(S)S) is a human DNA repair-deficient disease with mild clinical manifestations. No neurological or developmental abnormalities or predisposition to cancer have been reported. In contrast, Cockayne syndrome (CS) patients exhibit severe developmental and neurological defects, in addition to photosensitivity. The cellular and biochemical responses of UV(S)S and CS cells to UV are indistinguishable, and result from defective transcription-coupled repair (TCR) of photoproducts in expressed genes. We propose that UV(S)S patients develop normally because they are proficient in repair of oxidative base damage. Consistent with our model, we show that Cockayne syndrome cells from complementation groups A and B (CS-A, CS-B) are more sensitive to treatment with hydrogen peroxide than wild type or UV(S)S cells. Using a host cell reactivation assay with plasmids containing UV-induced photoproducts, we find that expression of the plasmid-encoded lacZ gene is reduced in the TCR-deficient CS-B and UV(S)S cells. When the plasmids contain the oxidative base lesion thymine glycol, CS-B cells are defective in recovery of expression, whereas UV(S)S cells show levels of expression similar to those in wild type cells. 8-oxoguanine in the plasmids result in similarly defective host cell reactivation in CS-A and CS-B cells; abasic sites or single strand breaks in the plasmids cause similar decreases in expression in all the cell lines examined. Repair of thymine glycols in the lacZ gene was measured in plasmids extracted from transfected cells; removal of the lesions is efficient and without strand bias in all the cell lines tested.

    View details for DOI 10.1016/j.dnarep.2005.06.017

    View details for Web of Science ID 000234780000002

    View details for PubMedID 16129663

  • Comparative TFIIS-mediated transcript cleavage by mammalian RNA polymerase II arrested at a lesion in different transcription systems DNA REPAIR Kalogeraki, V. S., Tornaletti, S., Cooper, P. K., Hanawalt, P. C. 2005; 4 (10): 1075-1087

    Abstract

    Upon prolonged arrest at a cyclobutane pyrimidine dimer (CPD), RNAPII can reverse-translocate, misaligning the 3'-end of the RNA from its active site. Transcription factor SII (TFIIS) is required for cleavage of the disengaged 3'-end and restoration of its correct positioning. We have previously shown in vitro that when RNAPII is arrested at a CPD, TFIIS-induced cleavage results in shortened transcripts. Here, we hypothesized that the pattern of transcript cleavage does not depend solely upon TFIIS itself, but also on some other general transcription factors (GTFs) and/or their effects on RNAPII. To test this hypothesis we compared three in vitro transcription systems which differ with respect to the mode of initiation and the requirement for GTFs. The first consisted of RNAPII and GTFs from rat liver, and required a eukaryotic promoter for initiation. The other two supported transcription in the absence of any GTFs or promoter sequences. In each case, a CPD on the transcribed strand was a complete block for RNAPII translocation. However, the effect of TFIIS on transcript cleavage varied. In the promoter-initiated system, distinct transcripts up to about 20 nucleotides shorter than the uncleaved original one were produced. In the other two systems, the transcripts were degraded nearly completely. Introduction of GTFs partially interfered with cleavage, but failed to reproduce the pattern of transcript lengths observed with the promoter-initiated system. Our results suggest that the extent of TFIIS-mediated transcript cleavage is a well-orchestrated process, depending upon other factors (or their effects on RNAPII), in addition to TFIIS itself.

    View details for DOI 10.1016/j.dnarep.2005.05.007

    View details for Web of Science ID 000232429100004

    View details for PubMedID 16046193

  • Density matters: The semiconservative replication of DNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hanawalt, P. C. 2004; 101 (52): 17889-17894

    Abstract

    The semiconservative mode of DNA replication was originally documented through the classic density labeling experiments of Matthew Meselson and Franklin W. Stahl, as communicated to PNAS by Max Delbruck in May 1958. The ultimate value of their novel approach has extended far beyond the initial implications from that elegant study, through more than four decades of research on DNA replication, recombination, and repair. I provide here a short historical commentary and then an account of some developments in the field of DNA replication, which closely followed the Meselson-Stahl experiment. These developments include the application of density labeling to discover the repair replication of damaged DNA, a "nonconservative" mode of synthesis in which faulty sections of DNA are replaced.

    View details for DOI 10.1073/pnas.0407539101

    View details for Web of Science ID 000226102700003

    View details for PubMedID 15608066

  • Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cline, S. D., Riggins, J. N., Tornaletti, S., Marnett, L. J., Hanawalt, P. C. 2004; 101 (19): 7275-7280

    Abstract

    Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient removal of M(1)dG in active genes. Results presented here show that M(1)dG and its stable, exocyclic analog 1,N(2)-propanodeoxyguanine (PdG), arrest translocation of T7 RNAP and mammalian RNAPII when located in the transcribed strand of a DNA template. M(1)dG paired with thymine is exocyclic and poses a stronger block to transcription than the acyclic N(2)-(3-oxo-1-propenyl)-dG, formed upon cytosine-catalyzed opening of M(1)dG in duplex DNA. PdG is a complete block to RNAPII regardless of base pairing. The elongation factor TFIIS (SII) induces reversal and RNA transcript cleavage by RNAPII arrested at PdG. Thus, arrested RNAPII complexes may be stable at M(1)dG in cells and may resume transcription once the offending adduct is removed. The conclusion from this work is that malondialdehyde adducts in the transcribed strand of expressed genes are strong blocks to RNAPs and are targets for cellular transcription-coupled repair. If so, then M(1)dG, already known to be highly mutagenic in human cells, also may contribute to apoptosis in the developing tissues of individuals with Cockayne's syndrome, a hereditary disorder characterized by transcription-coupled repair deficiency.

    View details for DOI 10.1073/pnas.0402252101

    View details for Web of Science ID 000221559100015

    View details for PubMedID 15123825

  • Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II DNA REPAIR Silvia, T. A., Maeda, L. S., Kolodner, R. D., Hanawalt, P. C. 2004; 3 (5): 483-494

    Abstract

    8-Oxoguanine (8-oxoG) is a major oxidative lesion produced in DNA by normal cellular metabolism or after exposure to exogenous sources such as ionizing radiation. Persistence of this lesion in DNA causes G to T transversions, with deleterious consequences for the cell. As a result, several repair processes have evolved to remove this lesion from the genome. It has been reported that 8-oxoG is subject to transcription-coupled repair (TCR), a process dedicated to removal of lesions from transcribed strands of expressed genes. A current model assumes that RNA polymerase arrest at the site of the lesion is required for initiation of TCR. As a first step to understand how TCR of 8-oxoG occurs, we have studied the effect of 8-oxoG on transcription elongation by T7 RNA polymerase (T7 RNAP) and rat liver RNA polymerase II (RNAPII). We have utilized an in vitro transcription system with purified RNA polymerase and initiation factors, and substrates containing a single 8-oxoG in the transcribed or in the non-transcribed strand downstream of the T7 promoter or the Adenovirus major late promoter. We found that 8-oxoG only slightly inhibited T7 RNAP transcription, with a readthrough frequency of up to 95%. Similarly, this lesion only transiently blocked transcription by RNAPII. However, changes in nucleotide concentration affected the extent of RNAPII blockage at the 8-oxoG. When this lesion was positioned in the non-transcribed strand, complete lesion bypass was observed with either polymerase. Binding of the Saccharomyces cerevisiae MSH2-MSH6 complex to 8-oxoG containing substrates did not increase the frequency of RNAPII arrest at the site of the lesion, suggesting that this complex was displaced by the elongating polymerase. These results are discussed in the context of possible models for TCR.

    View details for DOI 10.1016/j.dnarep.2004.01.003

    View details for Web of Science ID 000221156400005

    View details for PubMedID 15084310

  • Functional characterization of global genomic DNA repair and its implications for cancer MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH Hanawalt, P. C., Ford, J. A., Lloyd, D. R. 2003; 544 (2-3): 107-114

    Abstract

    The most versatile cellular pathway for dealing with a large variety of structurally-unrelated DNA alterations is nucleotide excision repair (NER). Most genomic damage, if not repaired, may contribute to mutagenesis and carcinogenesis, as well as to cellular lethality. There are two subpathways of NER, termed global genomic repair (GGR) and transcription-coupled repair (TCR); While GGR deals with all repairable lesions throughout the genome, TCR is selective for the transcribed DNA strand in expressed genes. Proteins involved in the initial recognition of lesions for GGR as well as for TCR (i.e. RNA polymerase) may sometimes initiate gratuitous repair events in undamaged DNA. However, the damage recognition enzymes for GGR are normally maintained at very low levels unless the cells are genomically stressed. Following UV irradiation in human fibroblasts the efficiency of GGR is upregulated through activation of the p53 tumor suppressor gene. The transactivation role of p53 includes control of expression of the genes, XPC and XPE, which are implicated in GGR but not TCR. These inducible responses are essential for the efficient repair of the most prominent lesion produced by UV, the cyclobutane pyrimidine dimer (CPD). They are also clinically relevant, as we have shown them to operate upon chemical carcinogen DNA damage at levels to which humans are environmentally exposed (e.g. through smoking). Thus, for benzo(a)pyrene (at 10-50 adducts per 10(8) nucleotides) repair was essentially complete within 1 day in p53(+/+) human fibroblasts while no repair was detected within 3 days in p53(-/-) cells. The levels of all four DNA adducts formed by benzo(g)chrysene, also exhibited p53-dependent control in human fibroblasts. However, unlike humans most rodent tissues are deficient in the p53-dependent GGR pathway. Since rodents are used as surrogates for humans in environmental cancer risk assessment it is very important that we determine how they differ from humans with respect to DNA repair and oncogenic responses to environmental genotoxins.

    View details for DOI 10.1016/j.mrrev.2003.06.002

    View details for Web of Science ID 000187353400005

    View details for PubMedID 14644313

  • Four decades of DNA repair: from early insights to current perspectives BIOCHIMIE Hanawalt, P. C. 2003; 85 (11): 1043-1052

    Abstract

    A brief history of the evolution of the DNA repair field over the past four decades is presented, as documented through the Proceedings from a selected series of five scientific meetings, beginning with the 1965 Radiation Microbiology Conference, held at the University of Chicago with only 40 participants, and extending through the 1988 UCLA Symposium on "Mechanisms and Consequences of DNA Damage Processing", convened in Taos, New Mexico, with over 400 participants. The published proceedings and recorded discussions from these early conferences contain notable insights, of which many have turned out to be remarkably clairvoyant while others must be reevaluated in light of recent discoveries and developments in the field.

    View details for DOI 10.1016/j.biochi.2003.11.007

    View details for Web of Science ID 000188616000002

    View details for PubMedID 14726012

  • Behavior of T7 RNA polymerase and mammalian RNA polymerase II at site-specific cisplatin adducts in the template DNA JOURNAL OF BIOLOGICAL CHEMISTRY Tornaletti, S., Patrick, S. M., Turchi, J. J., Hanawalt, P. C. 2003; 278 (37): 35791-35797

    Abstract

    Transcription-coupled DNA repair is dedicated to the removal of DNA lesions from transcribed strands of expressed genes. RNA polymerase arrest at a lesion has been proposed as a sensitive signal for recruitment of repair enzymes to the lesion site. To understand how initiation of transcription-coupled repair may occur, we have characterized the properties of the transcription complex when it encounters a lesion in its path. Here we have compared the effect of cisplatin-induced intrastrand cross-links on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We found that a single cisplatin 1,2-d(GG) intrastrand cross-link or a single cisplatin 1,3-d(GTG) intrastrand cross-link is a strong block to both polymerases. Furthermore, the efficiency of the block at a cisplatin 1,2-d(GG) intrastrand cross-link was similar in several different nucleotide sequence contexts. Interestingly, some blockage was also observed when the single cisplatin 1,3-d(GTG) intrastrand cross-link was located in the non-transcribed strand. Transcription complexes arrested at the cisplatin adducts were substrates for the transcript cleavage reaction mediated by the elongation factor TFIIS, indicating that the RNA polymerase II complexes arrested at these lesions are not released from template DNA. Addition of TFIIS yielded a population of transcripts up to 30 nucleotides shorter than those arrested at the lesion. In the presence of nucleoside triphosphates, these shortened transcripts could be re-elongated up to the site of the lesion, indicating that the arrested complexes are stable and competent to resume elongation. These results show that cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression, and they support current models of transcription arrest and initiation of transcription-coupled repair.

    View details for DOI 10.1074/jbc.M305394200

    View details for Web of Science ID 000185164400127

    View details for PubMedID 12829693

  • Transcription arrest at a lesion in the transcribed DNA strand in vitro is not affected by a nearby lesion in the opposite strand JOURNAL OF BIOLOGICAL CHEMISTRY Kalogeraki, V. S., Tornaletti, S., Hanawalt, P. C. 2003; 278 (21): 19558-19564

    Abstract

    Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA. CPDs are repaired by the nucleotide excision repair pathway. Additionally, they are subject to transcription-coupled DNA repair. In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion. Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases. Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand. Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls. Using an in vitro transcription system with purified T7 RNA polymerase (T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro. We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS. The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex. Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.

    View details for DOI 10.1074/jbc.M301060200

    View details for Web of Science ID 000182932200107

    View details for PubMedID 12646562

  • Who's on first in the cellular response to DNA damage? NATURE REVIEWS MOLECULAR CELL BIOLOGY Cline, S. D., Hanawalt, P. C. 2003; 4 (5): 361-372

    Abstract

    Cellular DNA-repair pathways involve proteins that have roles in other DNA-metabolic processes, as well as those that are dedicated to damage removal. Several proteins, which have diverse functions and are not known to have roles in DNA repair, also associate with damaged DNA. These newly discovered interactions could either facilitate or hinder the recognition of DNA damage, and so they could have important effects on DNA repair and genetic integrity. The outcome for the cell, and ultimately for the organism, might depend on which proteins arrive first at sites of DNA damage.

    View details for DOI 10.1038/nrm1101

    View details for Web of Science ID 000182687000017

    View details for PubMedID 12728270

  • When parsimony backfires: neglecting DNA repair may doom neurons in Alzheimer's disease BIOESSAYS Nouspikel, T., Hanawalt, P. C. 2003; 25 (2): 168-173

    Abstract

    Taking advantage of the fact that they need not replicate their DNA, terminally differentiated neurons only repair their expressed genes and largely dispense with the burden of removing damage from most of their genome. However, they may pay a heavy price for this laxity if unforeseen circumstances, such as a pathological condition like Alzheimer's disease, cause them to re-enter the cell cycle. The lifetime accumulation of unrepaired lesions in the silent genes of neurons is likely to be significant and may result in aborting the mitotic process and triggering cell death if the cells attempt to express these dormant genes and resume DNA replication.

    View details for DOI 10.1002/bies.10227

    View details for Web of Science ID 000180695000011

    View details for PubMedID 12539243

  • Subpathways of nucleotide excision repair and their regulation ONCOGENE Hanawalt, P. C. 2002; 21 (58): 8949-8956

    Abstract

    Nucleotide excision repair provides an important cellular defense against a large variety of structurally unrelated DNA alterations. Most of these alterations, if unrepaired, may contribute to mutagenesis, oncogenesis, and developmental abnormalities, as well as cellular lethality. There are two subpathways of nucleotide excision repair; global genomic repair (GGR) and transcription coupled repair (TCR), that is selective for the transcribed DNA strand in expressed genes. Some of the proteins involved in the recognition of DNA damage (including RNA polymerase) are also responsive to natural variations in the secondary structural features of DNA. Gratuitous repair events in undamaged DNA might then contribute to genomic instability. However, damage recognition enzymes for GGR are normally maintained at very low levels unless the cells are genomically stressed. GGR is controlled through the SOS stress response in E. coli and through the activated p53 tumor suppressor in human cells. These inducible responses in human cells are important, as they have been shown to operate upon chemical carcinogen DNA damage at levels to which humans are environmentally exposed. Interestingly, most rodent tissues are deficient in the p53-dependent GGR pathway. Since rodents are used as surrogates for environmental cancer risk assessment, it is essential that we understand how they differ from humans with respect to DNA repair and oncogenic responses to environmental genotoxins. In the case of terminally differentiated mammalian cells, a new paradigm has appeared in which GGR is attenuated but both strands of expressed genes are repaired efficiently.

    View details for DOI 10.1038/sj.onc.1206096

    View details for Web of Science ID 000179889500006

    View details for PubMedID 12483511

  • p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts CANCER RESEARCH Lloyd, D. R., Hanawalt, P. C. 2002; 62 (18): 5288-5294

    Abstract

    Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the maximum adduct levels achieved. The fact that this p53-dependent effect is noted at levels of DNA adducts that are commonly found in human tissues (i.e., <100 adducts/10(8) nucleotides) because of environmental factors such as smoking is particularly significant with respect to human carcinogenesis related to environmental exposure.

    View details for Web of Science ID 000178066400028

    View details for PubMedID 12234998

  • DNA repair in terminally differentiated cells DNA REPAIR Nouspikel, T., Hanawalt, P. C. 2002; 1 (1): 59-75

    Abstract

    Terminally differentiated cells do not replicate their genomic DNA, and could therefore dispense with the task of removing DNA damage from the non-essential bulk of their genome, as long as they are able to maintain the integrity of the genes that must be expressed. There is increasing experimental evidence that this is indeed the case, at least for some repair pathways such as nucleotide excision repair (NER). In this review, we examine a number of terminally differentiated cell systems in which it has been demonstrated that DNA repair is attenuated at the global genome level, but maintained in expressed genes. How these cells manage to repair transcribed genes is not yet fully elucidated, but there are indications that the transcription-coupled repair (TCR) pathway could maintain integrity of the transcribed strand (TS) in the active genes. We have observed in neurons that the non-transcribed strand (NTS) of active genes is also well repaired, a phenomenon that we have named differentiation-associated repair (DAR). It is conceivable that DAR is necessary to maintain the integrity of the template strand that is needed by TCR to complete the repair of lesions in the TS of essential expressed genes with high fidelity.

    View details for Web of Science ID 000182550600004

    View details for PubMedID 12509297

  • Effect of thymine glycol on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II JOURNAL OF BIOLOGICAL CHEMISTRY Tornaletti, S., Maeda, L. S., Lloyd, D. R., Reines, D., Hanawalt, P. C. 2001; 276 (48): 45367-45371

    Abstract

    Thymine glycols are formed in DNA by exposure to ionizing radiation or oxidative stress. Although these lesions are repaired by the base excision repair pathway, they have been shown also to be subject to transcription-coupled repair. A current model for transcription-coupled repair proposes that RNA polymerase II arrested at a DNA lesion provides a signal for recruitment of the repair enzymes to the lesion site. Here we report the effect of thymine glycol on transcription elongation by T7 RNA polymerase and RNA polymerase II from rat liver. DNA substrates containing a single thymine glycol located either in the transcribed or nontranscribed strand were used to carry out in vitro transcription. We found that thymine glycol in the transcribed strand blocked transcription elongation by T7 RNA polymerase approximately 50% of the time but did not block RNA polymerase II. Thymine glycol in the nontranscribed strand did not affect transcription by either polymerase. These results suggest that arrest of RNA polymerase elongation by thymine glycol is not necessary for transcription-coupled repair of this lesion. Additional factors that recognize and bind thymine glycol in DNA may be required to ensure RNA polymerase arrest and the initiation of transcription-coupled repair in vivo.

    View details for Web of Science ID 000172406700132

    View details for PubMedID 11571287

  • Spatially localized generation of nucleotide sequence-specific DNA damage PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Oh, D. H., King, B. A., Boxer, S. G., Hanawalt, P. C. 2001; 98 (20): 11271-11276

    Abstract

    Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues.

    View details for Web of Science ID 000171237100054

    View details for PubMedID 11572980

  • The SOS-dependent upregulation of uvrD is not required for efficient nucleotide excision repair of ultraviolet light induced DNA photoproducts in Escherichia coli MUTATION RESEARCH-DNA REPAIR Crowley, D. J., Hanawalt, P. C. 2001; 485 (4): 319-329

    Abstract

    We have shown previously that induction of the SOS response is required for efficient nucleotide excision repair (NER) of the major ultraviolet light (UV) induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), but not for repair of 6-4 photoproducts (6-4PP) or for transcription-coupled repair of CPDs [1]. We have proposed that the upregulation of cellular NER capacity occurs in the early stages of the SOS response and enhances the rate of repair of the abundant yet poorly recognized genomic CPDs. The expression of three NER genes, uvrA, uvrB, and uvrD, is upregulated as part of the SOS response. UvrD differs from the others in that it is not involved in lesion recognition but rather in promoting the post-incision steps of NER, including turnover of the UvrBC incision complex. Since uvrC is not induced during the SOS response, its turnover would seem to be of great importance in promoting efficient NER. Here we show that the constitutive level of UvrD is adequate for carrying out efficient NER of both CPDs and 6-4PPs. Thus, the upregulation of uvrA and uvrB genes during the SOS response is sufficient for inducible NER of CPDs. We also show that cells with a limited NER capacity, in this case due to deletion of the uvrD gene, repair 6-4PPs but cannot perform transcription-coupled repair of CPDs, indicating that the 6-4PP is a better substrate for NER than is a CPD targeted for transcription-coupled repair.

    View details for Web of Science ID 000168632600004

    View details for PubMedID 11585364

  • Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli GENETICS Courcelle, J., Khodursky, A., Peter, B., Brown, P. O., Hanawalt, P. C. 2001; 158 (1): 41-64

    Abstract

    The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.

    View details for Web of Science ID 000168637000005

    View details for PubMedID 11333217

  • Controlling the efficiency of excision repair MUTATION RESEARCH-DNA REPAIR Hanawalt, P. C. 2001; 485 (1): 3-13

    Abstract

    The early studies are recounted, that led to the discovery of the ubiquitous process of DNA excision repair, followed by a review of the pathways of transcription-coupled repair (TCR) and global genomic nucleotide excision repair (GGR). Repair replication of damaged DNA in UV-irradiated bacteria was discovered through the use of 5-bromouracil to density-label newly synthesized DNA. This assay was then used in human cells to validate the phenomenon of unscheduled DNA synthesis as a measure of excision repair and to elucidate the first example of a DNA repair disorder, xeroderma pigmentosum. Features of the TCR pathway (that is defective in Cockayne syndrome (CS)) include the possibility of "gratuitous TCR" at transcription pause sites in undamaged DNA. The GGR pathway is shown to be controlled through the SOS stress response in E. coli and through the activated product of the p53 tumor suppressor gene in human cells. These regulatory systems particularly affect the efficiency of repair of the predominant UV-induced photoproduct, the cyclobutane pyrimidine dimer, as well as that of chemical carcinogen adducts, such as benzo(a)pyrene diol-epoxide. Rodent cells (typically lacking the p53-controlled GGR pathway) and tumor virus infected human cells (in which p53 function is abrogated) are unable to carry out efficient GGR of some lesions. Therefore, caution should be exercised in the interpretation of results from such systems for risk assessment in genetic toxicology. Many problems in excision repair remain to be solved, including the mechanism of scanning the DNA for lesions and the subcellular localization of the repair factories. Also there are persisting questions regarding the multiple options of repair, recombination, and translesion synthesis when replication forks encounter lesions in the template DNA. That is where the field of DNA excision repair began four decades ago with studies on the recovery of DNA synthesis in UV-irradiated bacteria.

    View details for Web of Science ID 000167023500002

    View details for PubMedID 11341989

  • Revisiting the rodent repairadox ENVIRONMENTAL AND MOLECULAR MUTAGENESIS Hanawalt, P. C. 2001; 38 (2-3): 89-96

    Abstract

    Cultured rodent and human cells typically display similar clonal survival characteristics following exposure to ultraviolet light (UV). However, compared to human cells, cultured cells from mice, rats, and hamsters are generally deficient in excision repair of the most prominent DNA lesion produced by UV, the cyclobutane pyrimidine dimer. In light of recent studies on the control of nucleotide excision repair, we are beginning to understand the basis for this so-called "repairadox." The resolution of this issue is important because rodents are so widely employed as surrogates for humans in genetic toxicology. This article will review the evolution in our understanding of rodent DNA repair and will also "revisit" my early association with my graduate mentor and esteemed colleague, Dick Setlow, in his honor upon the attainment of his 80th birthday.

    View details for Web of Science ID 000172017400002

    View details for PubMedID 11746740

  • Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides PHOTOCHEMISTRY AND PHOTOBIOLOGY Oh, D. H., Hanawalt, P. C. 2000; 72 (3): 298-307

    Abstract

    Triple helix-forming oligonucleotides conjugated to a psoralen (psoTFO) have been designed to bind to three distinct purine-rich sequences within the human interstitial collagenase (MMP1) gene. Gel mobility shift assays indicate that these psoTFO bind to and photoreact with model target DNA sequences following ultraviolet A (UVA) irradiation. The dissociation constants for binding of the psoTFO to their targets range from 0.3 to 4 microM. Psoralen monoadducts with the purine-rich target strand and interstrand crosslinks are efficiently formed on targets containing either 5'-ApT-3' or 5'-TpA-3' sequences adjacent to the TFO binding sequence. The dependence of adduct formation on UVA dose has provided quantitative estimates of the overall rate constants for psoralen monoadduct and crosslink formation in the presence of a TFO. When psoralen is tethered to a TFO, the rate of monoadduct formation exceeds that of crosslinking for all sequences studied. This contrasts with the relatively low rate of monoadduct formation that has been reported for free psoralens, suggesting that the bound TFO facilitates the initial photochemistry that generates monoadducts, but does not significantly affect interstrand crosslink formation. psoTFO and UVA treatment inhibit DNA cleavage by a restriction endonuclease when the psoralen covalently reacts directly at the endonuclease site. The particular TFO studied do not completely inhibit endonuclease activity when they are noncovalently bound or when the covalent psoralen adduct does not coincide with the endonuclease site. Our findings confirm that TFO are capable of directing psoralen photoadducts to specific DNA targets and suggest that TFO can significantly modulate psoralen photoreactivity and DNA-protein interactions.

    View details for Web of Science ID 000089212700004

    View details for PubMedID 10989598

  • Reduced global genomic repair of ultraviolet light-induced cyclobutane pyrimidine dimers in simian virus 40-transformed human cells MOLECULAR CARCINOGENESIS Bowman, K. K., Sicard, D. M., Ford, J. M., Hanawalt, P. C. 2000; 29 (1): 17-24

    Abstract

    The p53 tumor-suppressor gene has been implicated in the inducible activation of excision repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in human cells. Because the large T antigen (LTAg) of the simian virus 40 (SV40) binds p53 protein and can interfere with its function, it was of interest to study DNA repair in normal human fibroblasts that had been transformed by SV40 compared with that in their nontransformed parental counterparts and to determine whether such transformation attenuated global genomic repair (GGR) of CPDs. Three methods were used to measure GGR in UV-irradiated cells: (i) an immunoassay using monoclonal antibodies specific for CPDs or 6-4 photoproducts (6-4PPs), (ii) zone sedimentation in alkaline sucrose gradients to measure the average DNA strand size after specific nicking at CPD sites in duplex DNA with T4 endonuclease V (TEV), and (iii) Southern hybridization of TEV-treated DNA with strand-specific mRNA probes to assess removal of CPDs from either strand of a defined genetic sequence in an expressed gene. Whereas repair of 6-4PPs was very similar in paired SV40-transformed and primary fibroblasts, GGR of CPDs was significantly reduced in the SV40-transformed cells. In contrast, SV40 transformation did not appreciably affect the efficiency of transcription-coupled repair. These data support the hypothesis that SV40 transformation can result in reduced levels of GGR, most likely because of the inhibition of normal p53 function by LTAg.

    View details for Web of Science ID 000089690900003

    View details for PubMedID 11020243

  • Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis MOLECULAR CELL Tang, J. Y., Hwang, B. J., Ford, J. M., Hanawalt, P. C., Chu, G. 2000; 5 (4): 737-744

    Abstract

    UV-damaged DNA-binding activity (UV-DDB) is deficient in some xeroderma pigmentosum group E individuals due to mutation of the p48 gene, but its role in DNA repair has been obscure. We found that UV-DDB is also deficient in cell lines and primary tissues from rodents. Transfection of p48 conferred UV-DDB to hamster cells, and enhanced removal of cyclobutane pyrimidine dimers (CPDs) from genomic DNA and from the nontranscribed strand of an expressed gene. Expression of p48 suppressed UV-induced mutations arising from the nontranscribed strand, but had no effect on cellular UV sensitivity. These results define the role of p48 in DNA repair, demonstrate the importance of CPDs in mutagenesis, and suggest how rodent models can be improved to better reflect cancer susceptibility in humans.

    View details for Web of Science ID 000086790000014

    View details for PubMedID 10882109

  • Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair and modulation of repair gene expression MOLECULAR AND CELLULAR BIOLOGY Nouspikel, T., Hanawalt, P. C. 2000; 20 (5): 1562-1570

    Abstract

    Repair of UV-induced DNA lesions in terminally differentiated human hNT neurons was compared to that in their repair-proficient precursor NT2 cells. Global genome repair of (6-4)pyrimidine-pyrimidone photoproducts was significantly slower in hNT neurons than in the precursor cells, and repair of cyclobutane pyrimidine dimers (CPDs) was not detected in the hNT neurons. This deficiency in global genome repair did not appear to be due to denser chromatin structure in hNT neurons. By contrast, CPDs were removed efficiently from both strands of transcribed genes in hNT neurons, with the nontranscribed strand being repaired unexpectedly well. Correlated with these changes in repair during neuronal differentiation were modifications in the expression of several repair genes, in particular an up-regulation of the two structure-specific nucleases XPG and XPF/ERCC1. These results have implications for neuronal dysfunction and aging.

    View details for Web of Science ID 000085342200011

    View details for PubMedID 10669734

  • p53-dependent global genomic repair of benzo[a]pyrene-7,8-diol-9,10-epoxide adducts in human cells CANCER RESEARCH Lloyd, D. R., Hanawalt, P. C. 2000; 60 (3): 517-521

    Abstract

    The global genomic repair of DNA adducts formed by the human carcinogen (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) has been studied by 32P-postlabeling in human fibroblasts in which p53 expression can be regulated. At low BPDE adduct levels (10-50 adducts/10(8) nucleotides), repair was rapid and essentially complete within 24 h in p53+ cells, whereas no repair was detected within 72 h in similarly treated p53- cells. At 10-fold higher BPDE adduct levels, repair under both conditions was rapid up to 8 h, after which a low level of adducts persisted only in p53- cells. These results demonstrate a dependence on p53 for the efficient repair of BPDE adducts at levels that are relevant to human environmental exposure and, thus, have significant implications for human carcinogenesis.

    View details for Web of Science ID 000085235600004

    View details for PubMedID 10676627

  • Regulation of nucleotide excision repair in bacteria and mammalian cells COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Hanawalt, P. C., Crowley, D. J., Ford, J. M., Ganesan, A. K., Lloyd, D. R., Nouspikel, T., Smith, C. A., Spivak, G., Tornaletti, S. 2000; 65: 183-191

    View details for Web of Science ID 000169676800019

    View details for PubMedID 12760032

  • Triple helix-forming oligonucleotides target psoralen adducts to specific chromosomal sequences in human cells NUCLEIC ACIDS RESEARCH Oh, D. H., Hanawalt, P. C. 1999; 27 (24): 4734-4742

    Abstract

    The ability to target photochemical adducts to specific genomic DNA sequences in cells is useful for studying DNA repair and mutagenesis in intact cells, and also as a potential mode of gene-specific therapy. Triple helix-forming DNA oligonucleotides linked to psoralen (psoTFOs) were designed to deliver UVA-induced psoralen photoadducts to two distinct sequences within the human interstitial collagenase gene. A primer extension assay demonstrated that the appropriate psoTFO selectively damages a collagenase cDNA target. Site-specific genomic psoTFO DNA adducts were detected by a single-strand ligation PCR assay. The adduct, formed at a single site by a psoTFO in purified genomic DNA, contrasted with the multiple sites that were damaged within the observed segment of the collagenase gene upon treatment with free psoralen and subsequent photoactivation. When treated with psoTFOs, both repair-deficient fibroblasts from xero- derma pigmentosum complementation group A and HT1080 fibrosarcoma cells exhibited site-specific DNA adducts following UVA irradiation. Addition of phorbol ester, a transcriptional activator of the collagenase gene, to xeroderma pigmentosum cells did not detectably alter the initial levels of damage produced by psoTFOs, suggesting that further stimulation of transcription neither improves accessibility of psoTFOs to their targets nor enhances removal of non-covalently bound psoTFOs.

    View details for Web of Science ID 000084547200009

    View details for PubMedID 10572173

  • A phylogenomic study of DNA repair genes, proteins, and processes MUTATION RESEARCH-DNA REPAIR Eisen, J. A., Hanawalt, P. C. 1999; 435 (3): 171-213

    Abstract

    The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence.

    View details for Web of Science ID 000084300500001

    View details for PubMedID 10606811

  • RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli MOLECULAR AND GENERAL GENETICS Courcelle, J., Hanawalt, P. C. 1999; 262 (3): 543-551

    Abstract

    The accurate recovery of replication following DNA damage and repair is critical for the maintenance of genomic integrity. In Escherichia coli, the recovery of replication following UV-induced DNA damage is dependent upon several proteins in the recF pathway, including RecF, RecO, and RecR. Two other recF pathway proteins, the RecQ helicase and the RecJ exonuclease, have been shown to affect the sites and frequencies at which illegitimate rearrangements occur following UV-induced DNA damage, suggesting that they also may function during the recovery of replication. We show here that RecQ and RecJ process the nascent DNA at blocked replication forks prior to the resumption of DNA synthesis. The processing involves selective degradation of the nascent lagging DNA strand and it requires both RecQ and RecJ. We suggest that this processing may serve to lengthen the substrate that can be recognized and stabilized by the RecA protein at the replication fork, thereby helping to ensure the accurate recovery of replication after the obstructing lesion has been repaired.

    View details for Web of Science ID 000083839000018

    View details for PubMedID 10589843

  • Structural characterization of RNA polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the transcribed strand of template DNA JOURNAL OF BIOLOGICAL CHEMISTRY Tornaletti, S., Reines, D., Hanawalt, P. C. 1999; 274 (34): 24124-24130

    Abstract

    We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested at a naturally occurring elongation impediment. The footprint of RNAP II arrested at a CPD, using exonuclease III and T4 DNA polymerase's 3'-->5' exonuclease, covers approximately 35 base pairs and is asymmetrically located around the dimer. A similar footprint is observed when RNAP II is arrested at the human histone H3.3 arrest site. Addition of elongation factor SII to RNAP II arrested at a CPD produced shortened transcripts of discrete lengths up to 25 nucleotides shorter than those seen without SII. After addition of photolyase and exposure to visible light, some of the transcripts could be reelongated beyond the dimer, suggesting that SII-mediated transcript cleavage accompanied significant RNAP II backup, thereby providing access of the repair enzyme to the arresting CPD.

    View details for Web of Science ID 000082110900063

    View details for PubMedID 10446184

  • Expression and nucleotide excision repair of a UV-irradiated reporter gene in unirradiated human cells MUTATION RESEARCH-DNA REPAIR Ganesan, A. K., Hunt, J., Hanawalt, P. C. 1999; 433 (2): 117-126

    Abstract

    It has been suggested that reactivation of damaged reporter genes introduced into cultured mammalian cells reflects transcription-coupled nucleotide excision repair. To evaluate this possibility directly, we introduced a UV-irradiated shuttle vector, pCMV beta, into unirradiated human cells and compared expression of the reporter gene (lacZ) with repair of cyclobutane pyrimidine dimers (CPDs). Expression of the irradiated reporter gene was more UV resistant in XPC cells, which are deficient in global genome repair, than in CSB cells, which are deficient in transcription-coupled repair. These results are consistent with the idea that repair of the reporter gene is primarily dependent upon transcription-coupled repair. However, when the plasmid DNA was analyzed for removal of CPDs, no clear evidence was obtained for transcription-coupled repair either in XPC cells or in cells with normal repair capacity.

    View details for Web of Science ID 000079208100005

    View details for PubMedID 10102038

  • Recovery of DNA replication in UV-irradiated Escherichia coli requires both excision repair and RecF protein function JOURNAL OF BACTERIOLOGY Courcelle, J., Crowley, D. J., Hanawalt, P. C. 1999; 181 (3): 916-922

    Abstract

    After UV doses that disrupt DNA replication, the recovery of replication at replication forks in Escherichia coli requires a functional copy of the recF gene. In recF mutants, replication fails to recover and extensive degradation of the nascent DNA occurs, suggesting that recF function is needed to stabilize the disrupted replication forks and facilitate the process of recovery. We show here that the ability of recF to promote the recovery of replication requires that the disrupting lesions be removed. In the absence of excision repair, recF+ cells protect the nascent DNA at replication forks, but replication does not resume. The classical view is that recombination proteins operate in pathways that are independent from DNA repair, and therefore the functions of Rec proteins have been studied in repair-deficient cells. However, mutations in either uvr or recF result in failure to recover replication at UV doses from which wild-type cells recover efficiently, suggesting that recF and excision repair contribute to a common pathway in the recovery of replication.

    View details for Web of Science ID 000078390600028

    View details for PubMedID 9922256

  • Expression of the p48 xeroderma pigmentosum gene is p53-dependent and is involved in global genomic repair PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hwang, B. J., Ford, J. M., Hanawalt, P. C., Chu, G. 1999; 96 (2): 424-428

    Abstract

    In human cells, efficient global genomic repair of DNA damage induced by ultraviolet radiation requires the p53 tumor suppressor, but the mechanism has been unclear. The p48 gene is required for expression of an ultraviolet radiation-damaged DNA binding activity and is disrupted by mutations in the subset of xeroderma pigmentosum group E cells that lack this activity. Here, we show that p48 mRNA levels strongly depend on basal p53 expression and increase further after DNA damage in a p53-dependent manner. Furthermore, like p53(-/-) cells, xeroderma pigmentosum group E cells are deficient in global genomic repair. These results identify p48 as the link between p53 and the nucleotide excision repair apparatus.

    View details for Web of Science ID 000078189200023

    View details for PubMedID 9892649

  • Effect of DNA lesions on transcription elongation BIOCHIMIE Tornaletti, S., Hanawalt, P. C. 1999; 81 (1-2): 139-146

    Abstract

    Some types of damage to cellular DNA have been shown to interfere with the essential transactions of replication and transcription. Not only may the translocation of the polymerase be arrested at the site of the lesion but the bound protein may encumber recognition of the lesion by repair enzymes. In the case of transcription a subpathway of excision repair, termed transcription-coupled repair (TCR) has been shown to operate on lesions in the transcribed strands of expressed genes in bacteria, yeast, mammalian cells and a number of other organisms. Certain genes in mammalian cells (e.g., CSA and CSB) have been uniquely implicated in TCR while others (e.g., XPC-HR23 and XPE) have been shown to operate in the global genomic pathway of nucleotide excision repair, but not in TCR. In order to understand the mechanism of TCR it is important to learn how an RNA polymerase elongation complex interacts with a damaged DNA template. That relationship is explored for different lesions and different RNA polymerase systems in this article.

    View details for Web of Science ID 000079411400015

    View details for PubMedID 10214918

  • Induction of the SOS response increases the efficiency of global nucleotide excision repair of cyclobutane pyrimidine dimers, but not 6-4 photoproducts, in UV-irradiated Escherichia coli JOURNAL OF BACTERIOLOGY Crowley, D. J., Hanawalt, P. C. 1998; 180 (13): 3345-3352

    Abstract

    Nucleotide excision repair (NER) is responsible for the removal of a variety of lesions from damaged DNA and proceeds through two subpathways, global repair and transcription-coupled repair. In Escherichia coli, both subpathways require UvrA and UvrB, which are induced following DNA damage as part of the SOS response. We found that elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampin reduced the efficiency of global repair of the major UV-induced lesion, the cyclobutane pyrimidine dimer (CPD), but had no effect on the global repair of 6-4 photoproducts. Mutants in which the SOS response was constitutively derepressed repaired CPDs more rapidly than did wild-type cells, and this rate was not affected by rifampin. Transcription-coupled repair of CPDs occurred in the absence of SOS induction but was undetectable when the response was expressed constitutively. These results suggest that damage-inducible synthesis of UvrA and UvrB is necessary for efficient repair of CPDs and that the levels of these proteins determine the rate of NER of UV photoproducts. We compare our findings with recent data from eukaryotic systems and suggest that damage-inducible stress responses are generally critical for efficient global repair of certain types of genomic damage.

    View details for Web of Science ID 000074416700011

    View details for PubMedID 9642186

  • Genomic instability: environmental invasion and the enemies within MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS Hanawalt, P. C. 1998; 400 (1-2): 117-125

    Abstract

    Deleterious alterations in cellular DNA result from endogenous sources of damage, as well as from external radiations and genotoxic chemicals in the environment. Although it is often difficult to ascertain the relative contributions to biological endpoints from endogenous vs. environmental sources of genomic instability, such determinations are highly relevant to risk estimates based upon perceived toxic levels of environmental agents. Of particular concern are the DNA lesions caused by reactive oxygen species that are generated both as a byproduct of oxidative metabolism and as a consequence of exposure to ionizing radiation and some other toxicants. We need to better understand the sequence of biochemical events that occurs between the initial formation of a DNA lesion and the biological outcome. These events may include transcription, replication, and cell cycle regulation, as well as DNA repair. Heterogeneity in the intragenomic distribution of lesions and their repair must also be taken into account. Expressed genes are unusually susceptible to alteration by some agents, and preferential repair of some lesions is targeted to transcribed DNA strands. An arrested RNA polymerase at a lesion may block access of repair enzymes, and it may also serve as a signal for upregulation of repair enzymes, cell cycle arrest and/or apoptosis. Our current understanding of the role of transcription in lesion processing and biological outcomes will be summarized, with particular emphasis upon the information gained from characterization of human genetic diseases expressing defects in the processing of damaged DNA. In some cases, the clinical features of these diseases might be understood in terms of deficiencies in the repair of lesions that arrest transcription.

    View details for Web of Science ID 000075548700012

    View details for PubMedID 9685605

  • Human fibroblasts expressing the human papillomavirus E6 gene are deficient in global genomic nucleotide excision repair and sensitive to ultraviolet irradiation CANCER RESEARCH Ford, J. M., Baron, E. L., Hanawalt, P. C. 1998; 58 (4): 599-603

    Abstract

    We investigated the role of wild-type p53 activity in modulating nucleotide excision repair after UV irradiation in normal and p53-deficient primary human fibroblasts created by expression of the human papillomavirus 16 E6 gene. Compared with parental cells, the E6-expressing fibroblasts were deficient in global genomic repair of both UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts but exhibited normal transcription-coupled repair. The E6-expressing cells were also more sensitive than their parental counterparts to UV irradiation and displayed similar levels of UV-induced apoptosis. These results suggest that disruption of wild-type p53 function by E6 expression results in selective loss of p53-dependent global genomic nucleotide excision repair, but not UV-induced apoptosis, leading to enhanced UV sensitivity.

    View details for Web of Science ID 000072025300007

    View details for PubMedID 9485006

  • Nucleotide sequence context effect of a cyclobutane pyrimidine dimer upon RNA polymerase II transcription JOURNAL OF BIOLOGICAL CHEMISTRY Tornaletti, S., Donahue, B. A., Reines, D., Hanawalt, P. C. 1997; 272 (50): 31719-31724

    Abstract

    We have studied the role of sequence context upon RNA polymerase II arrest by a cyclobutane pyrimidine dimer using an in vitro transcription system consisting of templates containing a specifically located cyclobutane pyrimidine dimer (CPD) and purified RNA polymerase II (RNAP II) and initiation factors. We selected a model sequence containing a well characterized site for RNAP II arrest in vitro, the human histone H3.3 gene arrest site. The 13-base pair core of the arrest sequence contains two runs of T in the nontranscribed strand that impose a bend in the DNA. We hypothesized that arrest of RNAP II might be affected by the presence of a CPD, based upon the observation that a CPD located at the center of a dA6.dT6 tract eliminates bending (Wang, C.-I., and Taylor, J.-S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9072-9076). We examined the normal H3.3 sequence and a mutant sequence containing a T --> G transversion, which reduces bending and efficiency of arrest. We show that a CPD in the transcribed strand at either of two locations in the arrest site is a potent block to transcription. However, a CPD in the nontranscribed strand only transiently pauses RNAP II. The CPD in concert with a mutation in the arrest site can reduce the extent of bending of the DNA and improve readthrough efficiency. These results demonstrate the potential importance of sequence context for the effect of CPDs within transcribed sequences.

    View details for Web of Science ID A1997YL41900074

    View details for PubMedID 9395515

  • Competent transcription initiation by RNA polymerase II in cell-free extracts from Xeroderma pigmentosum groups B and D in an optimized RNA transcription assay BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION Satoh, M. S., Hanawalt, P. C. 1997; 1354 (3): 241-251

    Abstract

    The human autosomal recessive disease, xeroderma pigmentosum (XP), can result from mutations in any one of seven genes, designated XPA through XPG. Of these, the XPB and XPD genes encode proteins that are subunits of a general transcription factor, TFIIH, involved in both nucleotide excision repair (NER) and initiation of mRNA transcription by RNA polymerase II. In humans, mutation of the XPB or XPD gene impairs NER, resulting in hyper-sensitivity to sunlight and greatly increased skin tumor formation. However, no transcription deficiency has been demonstrated in either XP-B or XP-D. We have employed an optimized cell-free RNA transcription assay to analyze transcription activity of XP-B and XP-D. Although the growth rate was normal, the XP-B and XP-D cells contained reduced amounts of TFIIH. Extracts prepared from XP-B and XP-D lymphoblastoid cells exhibited similar transcription activity from the adenovirus major late promoter when compared to that in extracts from normal cells. Thus, we conclude that the XP-B and XP-D lymphoblastoid cells do not have impaired RNA transcription activity. We consider the possible consequences of the reduced cellular content of TFIIH for the clinical symptoms in XP-B or XP-D patients, and discuss a 'conditional phenotype' that may involve an impairment of cellular function only under certain growth conditions.

    View details for Web of Science ID 000070986500007

    View details for PubMedID 9427533

  • Excision-repair patch lengths are similar for transcription-coupled repair and global genome repair in UV-irradiated human cells MUTATION RESEARCH-DNA REPAIR Bowman, K. K., Smith, C. A., Hanawalt, P. C. 1997; 385 (2): 95-105

    Abstract

    We have used the buoyant density shift method to measure excision-repair patch lengths in UV-irradiated repair-proficient human cells and in primary fibroblasts belonging to xeroderma pigmentosum complementation group C (XP-C), in which excision repair of UV-induced photoproducts is dependent upon transcription. The patch size was found to be about 30 nucleotides for both cell types. This agrees with the size of the DNA fragments excised in vitro by the dual incisions of the structure-specific nucleases XPG and ERCC1-XPF. We conclude that the XPC protein is not required to target the excision nucleases to sites of DNA cleavage in transcribed strands of expressed genes or to protect the newly incised DNA from further processing by exonucleases.

    View details for Web of Science ID 000071393900002

    View details for PubMedID 9447231

  • Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts JOURNAL OF BIOLOGICAL CHEMISTRY Ford, J. M., Hanawalt, P. C. 1997; 272 (44): 28073-28080

    Abstract

    We have shown previously that Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in the removal of UV-induced cyclobutane pyrimidine dimers from genomic DNA, but still proficient in the transcription-coupled repair pathway (Ford, J. M., and Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8876-8880). We have now utilized monoclonal antibodies specific for cyclobutane pyrimidine dimers or 6-4 photoproducts, respectively, to measure their repair in UV-irradiated human fibroblasts. Cells homozygous for p53 mutations were deficient in the repair of both photoproducts, whereas cells heterozygous for mutant p53 exhibited normal repair of 6-4 photoproducts, but decreased initial rates of removal of cyclobutane pyrimidine dimers, compared with normal cells. The specificity of the effect of wild-type p53 on nucleotide excision repair was demonstrated in a p53 homozygous mutant cell line containing a tetracycline-regulated wild-type p53 gene. Wild-type p53 expression and activity were suppressed in the presence of tetracycline, whereas withdrawal of tetracycline resulted in the induction of p53 expression, cell cycle checkpoint activation, and DNA damage-induced apoptosis. The regulated expression of wild-type p53 resulted in the recovery of normal levels of repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts in genomic DNA, but did not alter the transcription-coupled repair of cyclobutane pyrimidine dimers. Therefore, the wild-type p53 gene product is an important determinant of nucleotide excision repair activity in human cells.

    View details for Web of Science ID A1997YD47300087

    View details for PubMedID 9346961

  • recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Courcelle, J., CARSWELLCRUMPTON, C., Hanawalt, P. C. 1997; 94 (8): 3714-3719

    Abstract

    Escherichia coli containing a mutation in recF are hypersensitive to UV. However, they exhibit normal levels of conjugational or transductional recombination unless the major pathway (recBC) is defective. This implies that the UV sensitivity of recF mutants is not due to a defect in recombination such as occurs during conjugation or transduction. Here, we show that when replication is disrupted, at least two genes in the recF pathway, recF and recR, are required for the resumption of replication at DNA replication forks, and that in their absence, localized degradation occurs at the replication forks. Our observations support a model in which recF and recR are required to reassemble a replication holoenzyme at the site of a DNA replication fork. These results, when taken together with previous literature, suggest that the UV hypersensitivity of recF cells is due to an inability to resume replication at disrupted replication forks rather than to a defect in recombination. Current biochemical and genetic data on the conditions under which recF-mediated recombination occurs suggest that the recombinational intermediate also may mimic the structure of a disrupted replication fork.

    View details for Web of Science ID A1997WW81000044

    View details for PubMedID 9108043

  • Role of DNA excision repair gene defects in the etiology of cancer GENETIC INSTABILITY AND TUMORIGENESIS Ford, J. M., Hanawalt, P. C. 1997; 221: 47-70

    View details for Web of Science ID A1997BJ47J00005

    View details for PubMedID 8979440

  • TFIIH-mediated nucleotide excision repair and initiation of mRNA transcription in an optimized cell-free DNA repair and RNA transcription assay NUCLEIC ACIDS RESEARCH Satoh, M. S., Hanawalt, P. C. 1996; 24 (18): 3576-3582

    Abstract

    In mammalian cells, mRNA transcription is initiated with the aid of transcription initiation factors. Of these, TFIIH has also been shown to play an essential role in nucleotide excision repair (NER), which is a versatile biochemical pathway that corrects a broad range of DNA damage. Since the dual role of TFIIH is conserved among eukaryotes, including yeast and mammalian cells, the sharing of TFIIH between NER and RNA transcription initiation might provide some survival advantage. However, the functional relationship between NER and RNA transcription initiation through TFIIH is not yet understood. We have developed an optimized cell-free assay which allows us to analyze NER and RNA transcription under identical conditions. In this assay, NER did not compete with RNA transcription, probably because the extracts contained sufficient amounts of TFIIH to support both processes. Thus, NER can be considered functionally independent of RNA transcription initiation despite the fact that both processes use the same factor.

    View details for Web of Science ID A1996VK03200014

    View details for PubMedID 8836185

  • Recruitment of damaged DNA to the nuclear matrix in hamster cells following ultraviolet irradiation NUCLEIC ACIDS RESEARCH Koehler, D. R., Hanawalt, P. C. 1996; 24 (15): 2877-2884

    Abstract

    We examined the relationship between the nuclear matrix and DNA in the dihydrofolate reductase domain following irradiation of Chinese hamster cells with UV light. The fraction of matrix-bound DNA increased in transcribed and non-transcribed regions during a 3 h period after irradiation. However, no increase was observed with excision repair-deficient cells mutant for the ERCC1 gene. The major UV-induced lesion, the cyclobutane pyrimidine dimer, increased in frequency in the matrix-bound DNA 1 h after irradiation, in both transcribed and non-transcribed regions, but decreased subsequently. This phenomenon was also lacking in excision repair-deficient cells. These data demonstrate that recruitment of lesion-containing DNA to the nuclear matrix occurs following UV irradiation and suggest that this recruitment is dependent upon nucleotide excision repair. This is consistent with the concept of a 'repair factory' residing on the nuclear matrix at which excision repair occurs.

    View details for Web of Science ID A1996VB83200003

    View details for PubMedID 8760868

  • Mismatch repair mutants in yeast are not defective in transcription-coupled DNA repair of UV-induced DNA damage GENETICS Sweder, K. S., Verhage, R. A., Crowley, D. J., Crouse, G. F., Brouwer, J., Hanawalt, P. C. 1996; 143 (3): 1127-1135

    Abstract

    Transcription-coupled repair, the targeted repair of the transcribed strands of active genes, is defective in bacteria, yeast, and human cells carrying mutations in mfd, RAD26 and ERCC6, respectively. Other factors probably are also uniquely involved in transcription-repair coupling. Recently, a defect was described in transcription-coupled repair for Escherichia coli mismatch repair mutants and human tumor cell lines with mutations in mismatch repair genes. We examined removal of UV-induced DNA damage in yeast strains mutated in mismatch repair genes in an effort to confirm a defect in transcription-coupled repair in this system. In addition, we determined the contribution of the mismatch repair gene MSH2 to transcription-coupled repair in the absence of global genomic repair using rad7 delta mutants. We also determined whether the Rad26-independent transcription-coupled repair observed in rad26 delta and rad7 delta rad26 delta mutants depends on MSH2 by examining repair deficiencies of rad26 delta msh2 delta and rad7 delta rad26 delta msh2 delta mutants. We found no defects in transcription-coupled repair caused by mutations in the mismatch repair genes MSH2, MLH1, PMS1, and MSH3. Yeast appears to differ from bacteria and human cells in the capacity for transcription-coupled repair in a mismatch repair mutant background.

    View details for Web of Science ID A1996UU74200008

    View details for PubMedID 8807287

  • Effects of aminofluorene and acetylaminofluorene DNA adducts on transcriptional elongation by RNA polymerase II JOURNAL OF BIOLOGICAL CHEMISTRY Donahue, B. A., Fuchs, R. P., Reines, D., Hanawalt, P. C. 1996; 271 (18): 10588-10594

    Abstract

    A prominent model for the mechanism of transcription-coupled DNA repair proposes that an arrested RNA polymerase directs the nucleotide excision repair complex to the transcription-blocking lesion. The specific role for RNA polymerase II in this mechanism can be examined by comparing the extent of polymerase arrest with the extent of transcription-coupled repair for a specific DNA lesion. Previously we reported that a cyclobutane pyrimidine dimer that is repaired preferentially in transcribed genes is a strong block to transcript elongation by RNA pol II (Donahue, B.A., Yin, S., Taylor, J.-S., Reines, D., and Hanawalt, P. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8502-8506). Here we report the extent of RNA polymerase II arrest by the C-8 guanine DNA adduct formed by N-2-aminofluorene, a lesion that does not appear to be preferentially repaired. Templates for an in vitro transcription assay were constructed with either an N-2-aminofluorene adduct or the helix-distorting N-2-acetylaminofluorene adduct situated at a specific site downstream from the major late promoter of adenovirus. Consistent with the model for transcription-coupled repair, an aminofluorene adduct located on the transcribed strand was a weak pause site for RNA polymerase II. An acetylaminofluorene adduct located on the transcribed strand was an absolute block to transcriptional elongation. Either adduct located on the nontranscribed strand enhanced polymerase arrest at a nearby sequence-specific pause site.

    View details for Web of Science ID A1996UJ34200028

    View details for PubMedID 8631860

  • DNA repair deficiencies associated with mutations in genes encoding subunits of transcription initiation factor TFIIH in yeast NUCLEIC ACIDS RESEARCH Sweder, K. S., Chun, R., Mori, T., Hanawalt, P. C. 1996; 24 (8): 1540-1546

    Abstract

    Several proteins, including Rad3 and Rad25(Ssl2), are essential for nucleotide excision repair (NER) and function in the RNA polymerase II transcription initiation complex TFIIH. Mutations in genes encoding two other subunits of TFIIH, TFB1 and SSL1, result in UV sensitivity and have been shown to take part in NER in an in vitro system. However, a deficiency in global NER does not exclude the possibility that such repair-deficient mutants can perform transcription-coupled repair (TCR), as shown for xeroderma pigmentosum group C. To date, temperature-sensitive C-terminal truncations of Tfbl are the only TFIIH mutations that result in intermediate UV sensitivity, which might indicate a deficiency in either the global NER or TCR pathways. We have directly analyzed both TCR and global NER in these mutants. We found that ssl1, rad3 and tfb1 mutants, like rad25(ssl2-xp) mutants, are deficient in both the global NER and TCR pathways. Our results support the view that the mutations in any one of the genes encoding subunits of TFIIH result in deficiencies in both global and TCR pathways of NER. We suggest that when subunits of TFIIH are in limiting amounts, TCR may preclude global NER.

    View details for Web of Science ID A1996UH37000021

    View details for PubMedID 8628689

  • Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli JOURNAL OF BACTERIOLOGY Koehler, D. R., Courcelle, J., Hanawalt, P. C. 1996; 178 (5): 1347-1350

    Abstract

    We compared the removal of pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] and cyclobutane pyrimidine dimers (CPDs) from the genome of repair-proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion. We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2). The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs.

    View details for Web of Science ID A1996TX75200016

    View details for PubMedID 8631712

  • LI-FRAUMENI SYNDROME FIBROBLASTS HOMOZYGOUS FOR P53 MUTATIONS ARE DEFICIENT IN GLOBAL DNA-REPAIR BUT EXHIBIT NORMAL TRANSCRIPTION-COUPLED REPAIR AND ENHANCED UV RESISTANCE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ford, J. M., Hanawalt, P. C. 1995; 92 (19): 8876-8880

    Abstract

    We investigated whether mutations in the p53 tumor suppressor gene alter UV sensitivity and/or repair of UV-induced DNA damage in primary human skin fibroblasts from patients with Li-Fraumeni syndrome, heterozygous for mutations in one allele of the p53 gene (p53 wt/mut) and sublines expressing only mutant p53 (p53 mut). The p53 mut cells were more resistant than the p53 wt/mut cells to UV cytotoxicity and exhibited less UV-induced apoptosis. DNA repair analysis revealed reduced removal of cyclobutane pyrimidine dimers from overall genomic DNA in vivo in p53 mut cells compared with p53 wt/mut or normal cells. However, p53 mut cells retained the ability to preferentially repair damage in the transcribed strands of expressed genes (transcription-coupled repair). These results suggest that loss of p53 function may lead to greater genomic instability by reducing the efficiency of DNA repair but that cellular resistance to DNA-damaging agents may be enhanced through elimination of apoptosis.

    View details for Web of Science ID A1995RU75900070

    View details for PubMedID 7568035

  • EVOLUTION OF THE SNF2 FAMILY OF PROTEINS - SUBFAMILIES WITH DISTINCT SEQUENCES AND FUNCTIONS NUCLEIC ACIDS RESEARCH Eisen, J. A., Sweder, K. S., Hanawalt, P. C. 1995; 23 (14): 2715-2723

    Abstract

    The SNF2 family of proteins includes representatives from a variety of species with roles in cellular processes such as transcriptional regulation (e.g. MOT1, SNF2 and BRM), maintenance of chromosome stability during mitosis (e.g. lodestar) and various aspects of processing of DNA damage, including nucleotide excision repair (e.g. RAD16 and ERCC6), recombinational pathways (e.g. RAD54) and post-replication daughter strand gap repair (e.g. RAD5). This family also includes many proteins with no known function. To better characterize this family of proteins we have used molecular phylogenetic techniques to infer evolutionary relationships among the family members. We have divided the SNF2 family into multiple subfamilies, each of which represents what we propose to be a functionally and evolutionarily distinct group. We have then used the subfamily structure to predict the functions of some of the uncharacterized proteins in the SNF2 family. We discuss possible implications of this evolutionary analysis on the general properties and evolution of the SNF2 family.

    View details for Web of Science ID A1995RN26800022

    View details for PubMedID 7651832

  • DNA-REPAIR COMES OF AGE MUTATION RESEARCH-DNA REPAIR Hanawalt, P. C. 1995; 336 (2): 101-113

    View details for Web of Science ID A1995QL23700001

    View details for PubMedID 7885381

  • PREFERENTIAL REPAIR OF THE TRANSCRIBED DNA STRAND IN THE DIHYDROFOLATE-REDUCTASE GENE THROUGHOUT THE CELL-CYCLE IN UV-IRRADIATED HUMAN-CELLS MUTATION RESEARCH-DNA REPAIR Lommel, L., CARSWELLCRUMPTON, C., Hanawalt, P. C. 1995; 336 (2): 181-192

    Abstract

    We examined repair of UV-induced cyclobutane pyrimidine dimers (CPD) in each strand of the expressed dihydrofolate reductase gene in human cells in different phases of the cell cycle: G1, early S, middle S, late S, and G2/M. After 4 h of incubation, repair of the transcribed strand was substantially more efficient than repair of the non-transcribed strand in all phases. Furthermore, we observed no remarkable cell cycle-dependent differences in either the initial lesion frequency or the efficiency of repair of the transcribed strand. We conclude that transcription coupled repair operates generally and with high efficiency throughout the cell cycle.

    View details for Web of Science ID A1995QL23700009

    View details for PubMedID 7885388

  • DNA-REPAIR IN THE MYC AND FMS PROTOONCOGENES IN ULTRAVIOLET LIGHT-IRRADIATED HUMAN HL-60 PROMYELOCYTIC CELLS DURING DIFFERENTIATION CANCER RESEARCH Islas, A. L., Hanawalt, P. C. 1995; 55 (2): 336-341

    Abstract

    In order to better understand the role of transcription in cellular processing of damage in specific DNA sequences, we have used an in vitro differentiation system to modulate the activity of the MYC gene. When human HL60 promyelocytic cells differentiate in vitro, the transcriptional activity of the MYC gene is down-regulated. We have shown that in the expressed MYC gene, 56% of UV-induced cyclobutane pyrimidine dimers (CPDs) are removed within 18 h and the transcribed strand is selectively repaired. However, late in differentiation, when the MYC gene is maximally down-regulated, only 15% of the CPDs are removed within the same period. During early differentiation, the MYC gene is regulated by a block to transcription elongation at the 5' end of the first intron. Our results reveal no significant difference in the rate of CPD removal between the restriction fragments upstream and downstream of this elongation block. Furthermore, both strands of each fragment exhibit similar repair characteristics. In contrast, the constitutively expressed FMS gene exhibits proficient removal of CPD in both the differentiated and undifferentiated cells. Furthermore, the repair appears to be more proficient at the 5' end (exon 1) than in the 3' end of the gene about 35 kilobases downstream from exon 1. Since efficient repair of the active FMS gene is maintained in the differentiated cells the loss of repair competence seen in MYC is more likely associated with its reduced transcriptional activity than with a decrease in the overall repair capacity of the terminally differentiated cells.

    View details for Web of Science ID A1995QB80800024

    View details for PubMedID 7529133

  • TRANSCRIPTION-COUPLED REPAIR OF PSORALEN CROSS-LINKS BUT NOT MONOADDUCTS IN CHINESE-HAMSTER OVARY CELLS BIOCHEMISTRY Islas, A. L., Baker, F. J., Hanawalt, P. C. 1994; 33 (35): 10794-10799

    Abstract

    We have examined the rate and extent of removal of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linkable monoadducts and interstrand cross-links from restriction fragments within the amplicon containing the dihydrofolate reductase (DHFR) gene in the Chinese hamster ovary (CHO) cell line B11. The rate and extent of removal of HMT cross-links was significantly greater in an actively transcribed fragment than in a nontranscribed extragenic fragment of similar size. For the 5' half of the DHFR gene, approximately 80% of the HMT cross-links were removed in 8 h, in agreement with results reported by Vos and Wauthier [Vos, J. M., & Wauthier, E. L. (1991) Mol. Cell Biol. 11, 2245-2252, 1991]. However, few cross-links were removed in that period from the nontranscribed fragments, whose 5' end is approximately 7 kb downstream from the DHFR transcription unit and which includes a putative replication initiation site. Even after 24 h, only about 50% of the cross-links had been removed from this fragment. In contrast, both the rate and the extent of removal of cross-linkable HMT monoadducts were similar in the two fragments with 50% of the cross-linkable monoadducts removed in 24 h. Moreover, monoadducts formed in the bulk of the genome were removed in 24 h. Moreover, monoadducts formed in the bulk of the genome were removed at a slightly slower rate and to a lesser extent (30% in 24 hours) than those from either of these specific sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1994PF19000029

    View details for PubMedID 8075081

  • TRANSCRIPT CLEAVAGE BY RNA-POLYMERASE-II ARRESTED BY A CYCLOBUTANE PYRIMIDINE DIMER IN THE DNA-TEMPLATE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Donahue, B. A., Yin, S., Taylor, J. S., Reines, D., Hanawalt, P. C. 1994; 91 (18): 8502-8506

    Abstract

    A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcription-coupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SII (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SII induces nascent transcript cleavage by RNA polymerase II stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template.

    View details for Web of Science ID A1994PE38800040

    View details for PubMedID 8078911

  • PREFERENTIAL REPAIR OF ULTRAVIOLET LIGHT-INDUCED DNA-DAMAGE IN THE TRANSCRIBED STRAND OF THE HUMAN P53 GENE MOLECULAR CARCINOGENESIS Ford, J. M., Lommel, L., Hanawalt, P. C. 1994; 10 (2): 105-109

    Abstract

    Mutations in the p53 tumor suppressor gene have been found in most human tumors. Analyses of the spectrum of p53 mutations in certain tumor types have shown a bias for mutations originating from lesions presumed to be in the untranscribed strand of the gene. This implies strand specificity for the formation or repair of DNA damage. We measured the induction and repair of ultraviolet light-induced cyclobutane pyrimidine dimers (CPD) in each strand of the human p53 gene in a normal human lung fibroblast cell line using quantitative Southern hybridization. We found that the removal of CPD from the transcribed strand was more rapid than that from the untranscribed strand of this gene, although the frequency of CPD induction was similar in both strands. Preferential repair of the transcribed strand of the p53 gene may account for the mutational spectra of this gene in human tumors.

    View details for Web of Science ID A1994NV25200007

    View details for PubMedID 8031463

  • REPAIR AND TRANSCRIPTION - COLLISION OR COLLUSION CURRENT BIOLOGY Hanawalt, P. C., Donahue, B. A., Sweder, K. S. 1994; 4 (6): 518-521

    Abstract

    While some proteins have distinct responsibilities in both transcription and DNA repair, additional proteins are needed to couple these essential DNA transactions in expressed genes.

    View details for Web of Science ID A1994NU41000005

    View details for PubMedID 7864939

  • REMOVAL OF CYCLOBUTANE PYRIMIDINE DIMERS FROM A UV-IRRADIATED SHUTTLE VECTOR INTRODUCED INTO HUMAN-CELLS SOMATIC CELL AND MOLECULAR GENETICS Ganesan, A. K., Hanawalt, P. C. 1994; 20 (3): 233-242

    Abstract

    A shuttle vector (pZH-1) carrying the E. coli lacZ gene under control of the SV40 early promoter was irradiated with UV and introduced into repair-proficient or repair-deficient human cell lines. The expression of irradiated lacZ compared to unirradiated lacZ was greater in repair-proficient cells (HT-1080) than in repair-deficient cells (XP12RO-SV40) belonging to xeroderma pigmentosum complementation group A. To ascertain whether the expression of lacZ in the repair-proficient cells was correlated with the removal of cyclobutane pyrimidine dimers (CPDs), we purified DNA from the recipient cells and used the CPD-specific enzyme T4 endonuclease V to measure the frequency of CPDs remaining in the plasmid as a whole and in two restriction fragments derived from it. We found that removal of CPDs occurred in both fragments in the repair-proficient cells but not in the repair-deficient cells. Our results provide the first direct evidence for the removal of CPDs from UV irradiated plasmids introduced into human cells and support the notion that expression of the UV-damaged lacZ gene in repair-proficient human cells reflects the removal of transcription blocking lesions from the gene.

    View details for Web of Science ID A1994PJ61800008

    View details for PubMedID 7940023

  • REPAIR IN RIBOSOMAL-RNA GENES IS DEFICIENT IN XERODERMA-PIGMENTOSUM GROUP-C AND IN COCKAYNES-SYNDROME CELLS MUTATION RESEARCH Christians, F. C., Hanawalt, P. C. 1994; 323 (4): 179-187

    Abstract

    Previous studies have demonstrated transcription-coupled DNA repair in mammalian genes transcribed by RNA polymerase II but not in ribosomal RNA genes (rDNA), which are transcribed by RNA polymerase I. The removal of UV-induced cyclobutane pyrimidine dimers (CPD) from rDNA in repair-proficient human cells has been shown to be slow but detectable and apparently not coupled to transcription. We studied the induction and removal of CPD from rDNA in cultured cells from two repair-deficient human disorders. Primary xeroderma pigmentosum complementation group C (XP-C) cells, whether proliferating or nondividing, removed no CPD from either rDNA strand in 24 h post-UV, a result which supports earlier conclusions that XP-C cells lack the general, transcription-independent pathway of nucleotide excision repair. We also observed lower than normal repair of rDNA in Cockayne's syndrome (CS) cells from complementation groups A and B. In agreement with previous findings, the repair of both strands of the RNA polymerase II-transcribed dihydrofolate reductase gene was also deficient relative to that of normal cells. This strongly suggests that the defect in CS cells is not limited to a deficiency in a transcription-repair coupling factor. Rather, the defect may interfere with the ability of repair proteins to gain access to all expressed genes.

    View details for Web of Science ID A1994NE71200005

    View details for PubMedID 7512688

  • THE COOH TERMINUS OF SUPPRESSOR OF STEM-LOOP (SSL2/RAD25) IN YEAST IS ESSENTIAL FOR OVERALL GENOMIC EXCISION-REPAIR AND TRANSCRIPTION-COUPLED REPAIR JOURNAL OF BIOLOGICAL CHEMISTRY Sweder, K. S., Hanawalt, P. C. 1994; 269 (3): 1852-1857

    Abstract

    We examined several yeast strains with different mutations in the essential SSL2 (Suppressor of Stem Loop, also called RAD25) gene for their ability to remove cyclobutane pyrimidine dimers from expressed genes, and from the genome overall. The SSL2 protein has a high degree of amino acid sequence identity to the protein encoded by the human ERCC3 gene (Gulyas, K. D., and Donahue, T. F. (1992) Cell 69, 1031-0142). The mutant allele SSL2-XP encodes a protein resembling the mutated ERCC3 protein from UV-sensitive human cells belonging to xeroderma pigmentosum complementation group B and Cockayne's syndrome (CS) complementation group C (Weeda, G., van Ham, R. C. A., Vermeulen, W., Bootsma, D., van der Eb, A. J., and Hoeijmakers, J. H. J. (1990) Cell 62, 777-791; Gulyas and Donahue, 1992). The SSL2-XP allele confers UV sensitivity on yeast strain KG119. We found that the biochemical basis for the UV sensitivity of KG119 is a complete deficiency in the removal of cyclobutane pyrimidine dimers from the overall genome as well as a deficiency in transcription-coupled repair. This is the first analysis of the DNA repair defect responsible for the UV sensitivity of cells carrying the SSL2-XP allele, and it documents the similarity of the defect to that associated with XP-B/CS-C, and the difference between this defect and that in cells belonging to CS complementation groups A and B.

    View details for Web of Science ID A1994MR98800048

    View details for PubMedID 8294433

  • EVOLUTION OF CONCEPTS IN DNA-REPAIR ENVIRONMENTAL AND MOLECULAR MUTAGENESIS Hanawalt, P. C. 1994; 23: 78-85

    Abstract

    A short personalized history of the development of the field of DNA excision repair is presented, beginning with the early insights of radiation biologists and extending to the present-day convergence of the fields of DNA repair and transcription. It is becoming increasingly clear that excision repair is not merely an extraordinary scheme to help UV-exposed cells survive but rather one that operates upon a wide range of structural defects in DNA, some of which are due to environmental chemicals and others are a consequence of normal metabolic activities. It is an important challenge to researchers and risk assessors to determine the relative contributions to biological endpoints from endogenous events and the intrinsic instability of DNA as compared to exogenous environmental exposures. This should be one of the goals of the Environmental Mutagen Society as it embarks upon its second quarter-century.

    View details for Web of Science ID A1994NJ84400016

    View details for PubMedID 8162914

  • LACK OF TRANSCRIPTION-COUPLED REPAIR IN MAMMALIAN RIBOSOMAL-RNA GENES BIOCHEMISTRY Christians, F. C., Hanawalt, P. C. 1993; 32 (39): 10512-10518

    Abstract

    We studied the induction and removal of UV-induced cyclobutane pyrimidine dimers (CPDs) in the ribosomal RNA genes (rDNA) in cultured hamster and human cells. In these genes, which are transcribed by RNA polymerase I, we found no evidence for transcription-coupled repair. The induction of CPDs was heterogeneous in rDNA due to nucleotide sequence: it was lower on the transcribed strand than on the nontranscribed strand and slightly lower in the coding region than in the nontranscribed spacer. Nevertheless, no dramatic difference in CPD induction was observed between rDNA and the dihydrofolate reductase (DHFR) gene. In Chinese hamster ovary cells, we observed no removal of CPDs from either rDNA strand within 24 h after UV irradiation. In these experiments, we did observe efficient repair of the transcribed, but not the nontranscribed, strand of the DHFR gene, in agreement with published results. In human cells, repair of rDNA was observed, but it showed no strand preference and was slower than that reported for the genome overall. No significant differences in repair were observed between restriction fragments from transcribed and nontranscribed regions or between growth-arrested and proliferating human cells, with presumably different levels of transcription of rDNA. We conclude that the modest level of rDNA repair is accomplished by a transcription-independent repair system and that repair is impeded by the nucleolar compartmentalization of rDNA. We discuss the possibility that recombination, rather than repair, maintains the normal sequence of rDNA in mammalian cells.

    View details for Web of Science ID A1993MA66400030

    View details for PubMedID 8399197

  • CLOSE-FITTING SLEEVES - RECOGNITION OF STRUCTURAL DEFECTS IN DUPLEX DNA MUTATION RESEARCH Hanawalt, P. C. 1993; 289 (1): 7-15

    Abstract

    The first step in the ubiquitous cellular process of nucleotide excision-repair must involve the recognition of a lesion or structural distortion in DNA. This is followed by incision in the strand perceived as damaged; and then coordinated steps of local degradation and re-synthesis occur to replace the defective DNA segment with a new stretch of nucleotides, making use of the intact complementary strand as template. The repair patch is ultimately ligated at its 3' end to the contiguous preexisting DNA strand to restore the integrity of the normal DNA structure. Crucial to this repair scheme is the fact that the genome consists of double-stranded DNA, so that when one strand is damaged the information for its repair can, in principle, be recovered from the other strand. We will review a bit of the early speculation about the nature of the damage recognition step and then discuss the complexity of that event as we currently understand it. An important conceptual contribution to this field resulted from my collaboration with Robert Haynes in which we suggested that "the recognition step in the repair mechanism could be formally equivalent to threading the DNA through a close-fitting 'sleeve' which gauges the closeness-of-fit to the Watson-Crick structure" (Hanawalt and Haynes, 1965).

    View details for Web of Science ID A1993LV53600003

    View details for PubMedID 7689165

  • DIGESTION OF DAMAGED DNA BY THE T7 DNA POLYMERASE-EXONUCLEASE BIOCHEMICAL JOURNAL Koehler, D. R., Hanawalt, P. C. 1993; 293: 451-453

    Abstract

    We have investigated the 3'-5'-exonuclease activity of phage T7 DNA polymerase for its usefulness as an approach for the detection of lesions in DNA. Unlike the T4 DNA polymerase-exonuclease, which is commonly used to map the position and frequency of lesions in very small DNA fragments, T7 DNA polymerase-exonuclease is able to hydrolyse almost completely the large fragments from KpnI-restricted mammalian DNA. However, we found that the exonuclease was also able to hydrolyse DNA containing several kinds of lesions: cyclobutane pyrimidine dimers, thymine glycols, and mono-adducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen and 5'-methyl-isopsoralen. Modifications of the reaction conditions did not significantly alter the extent of hydrolysis. These properties distinguish the T7 DNA polymerase-exonuclease from the T4 DNA polymerase-exonuclease and make the T7 DNA polymerase-exonuclease unsuitable for detecting several types of lesions in DNA.

    View details for Web of Science ID A1993LP71300023

    View details for PubMedID 8343124

  • INCREASED UV RESISTANCE OF A XERODERMA-PIGMENTOSUM REVERTANT CELL-LINE IS CORRELATED WITH SELECTIVE REPAIR OF THE TRANSCRIBED STRAND OF AN EXPRESSED GENE MOLECULAR AND CELLULAR BIOLOGY Lommel, L., Hanawalt, P. C. 1993; 13 (2): 970-976

    Abstract

    A UV-resistant revertant (XP129) of a xeroderma pigmentosum group A cell line has been reported to be totally deficient in repair of cyclobutane pyrimidine dimers (CPDs) but proficient in repair of 6-4 photoproducts. This finding has been interpreted to mean that CPDs play no role in cell killing by UV. We have analyzed the fine structure of repair of CPDs in the dihydrofolate reductase gene in the revertant. In this essential, active gene, we observe that repair of the transcribed strand is as efficient as that in normal, repair-proficient human cells, but repair of the nontranscribed strand is not. Within 4 h after UV at 7.5 J/m2, over 50% of the CPDs were removed, and by 8 h, 80% of the CPDs were removed. In contrast, there was essentially no removal from the nontranscribed strand even by 24 h. Our results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs from the transcribed strands of expressed genes is essential for UV resistance.

    View details for Web of Science ID A1993KH79300027

    View details for PubMedID 8423816

  • PREFERENTIAL REPAIR OF CYCLOBUTANE PYRIMIDINE DIMERS IN THE TRANSCRIBED STRAND OF A GENE IN YEAST CHROMOSOMES AND PLASMIDS IS DEPENDENT ON TRANSCRIPTION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sweder, K. S., Hanawalt, P. C. 1992; 89 (22): 10696-10700

    Abstract

    While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia coli, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARS1 plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpb1-1, in the largest subunit of RNA polymerase II. When exponentially growing rpb1-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpb1-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpb1-1 cells was reduced to that in the nontranscribed strand. These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast.

    View details for Web of Science ID A1992JY87400028

    View details for PubMedID 1438266

  • INHIBITION OF TRANSCRIPTION AND STRAND-SPECIFIC DNA-REPAIR BY ALPHA-AMANITIN IN CHINESE-HAMSTER OVARY CELLS MUTATION RESEARCH Christians, F. C., Hanawalt, P. C. 1992; 274 (2): 93-101

    Abstract

    Recent studies have shown preferential repair of UV-induced cyclobutane pyrimidine dimers (CPD) in the transcribed strand of the expressed dihydrofolate reductase (DHFR) gene in human and rodent cells. We have tested the hypothesis that the strand-specific repair of such transcription-blocking lesions is dependent upon concurrent transcription. Chinese hamster ovary (CHO) B11 cells with an amplified DHFR gene were treated with alpha-amanitin before irradiation with UV (254 nm) and during post-irradiation incubation. Nuclear run-off analysis verified inhibition of transcription in the DHFR gene. CsCl density gradient analysis showed that alpha-amanitin at the levels used does not significantly interfere with overall genomic repair replication or semiconservative replication. However, we did observe a dramatic reduction in the removal of CPD from the transcribed strand in the 14 kb KpnI fragment within the DHFR gene in treated cells. We conclude that strand-specific repair of an active gene in CHO cells is dependent upon the activity of the transcribing RNA polymerase. Our results support the model that transcription complexes stalled at CPD signal the repair machinery to achieve efficient repair of the transcribed strand in active genes.

    View details for Web of Science ID A1992JC64900004

    View details for PubMedID 1378211

  • TRANSLESION DNA-SYNTHESIS IN THE DIHYDROFOLATE-REDUCTASE DOMAIN OF UV-IRRADIATED CHO CELLS BIOCHEMISTRY Spivak, G., Hanawalt, P. C. 1992; 31 (29): 6794-6800

    Abstract

    The studies that document the coupling of strand-specific DNA repair to transcription of active genes exclude replicated DNA from the analysis. Yet cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light (UV) persist in most of the genome in surviving Chinese hamster ovary (CHO) cells. The mechanisms that allow DNA replication to occur in the presence of damaged templates are poorly understood. We have investigated the distribution of CPD in the dihydrofolate reductase gene (DHFR) domain in replicated DNA. CHO B11 cells were incubated in the presence of BrdUrd after UV irradiation; the replicated DNA was separated from the unreplicated DNA by isopycnic sedimentation in CsCl, and then the parental and daughter strands were resolved in alkaline CsCl. We determined the fraction of a 14-kb KpnI fragment of the DHFR gene that was resistant to digestion by T4 endonuclease V, a CPD-specific enzyme. In both parental and unreplicated DNA, approximately 80% of the CPD were removed from the transcribed strands while approximately 20% were removed from the nontranscribed strands of DHFR within 24 h. In a 15-kb KpnI fragment that contains an origin of replication and is located approximately 15 kb downstream of DHFR, we found very low repair levels, whether it had been replicated or not. We detected no CPD in the daughter strands of either fragment analyzed. These results suggest that the replication forks can move through the damaged DNA in the absence of significant levels of repair or strand exchange and that the repair of CPD is not affected by replication in these cells.

    View details for Web of Science ID A1992JF65400021

    View details for PubMedID 1637815

  • LOCALIZED TORSIONAL TENSION IN THE DNA OF HUMAN-CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ljungman, M., Hanawalt, P. C. 1992; 89 (13): 6055-6059

    Abstract

    Torsional tension in DNA may be both a prerequisite for the efficient initiation of transcription and a consequence of the transcription process itself with the generation of positive torsional tension in front of the RNA polymerase and negative torsional tension behind it. To examine torsional tension in specific regions of genomic DNA in vivo, we developed an assay using photoactivated psoralen as a probe for unconstrained DNA superhelicity and x-rays as a means to relax DNA. Psoralen intercalates more readily into DNA underwound by negative torsional tension than into relaxed. DNA, and it can form interstrand DNA cross-links upon UVA irradiation. By comparing the amount of psoralen-induced DNA cross-links in cells irradiated with x-rays either before or after the psoralen treatment, we examined the topological state of the DNA in specific regions of the genome in cultured human 6A3 cells. We found that although no net torsional tension was detected in the bulk of the genome, localized tension was prominent in the DNA of two active genes. Negative torsional tension was found in the 5' end of the amplified dihydrofolate reductase gene and in a region near the 5' end of the 45S rRNA transcription unit, whereas a low level of positive torsional tension was found in a region near the 3' end of the dihydrofolate reductase gene. These results document an intragenomic heterogeneity of DNA torsional tension and lend support to the twin supercoiled domain model for transcription in the genome of intact human cells.

    View details for Web of Science ID A1992JC86800073

    View details for PubMedID 1631091

  • EFFICIENT PROTECTION AGAINST OXIDATIVE DNA DAMAGE IN CHROMATIN MOLECULAR CARCINOGENESIS Ljungman, M., Hanawalt, P. C. 1992; 5 (4): 264-269

    Abstract

    The role of histones and higher order chromatin structures in protecting against oxidative DNA damage was investigated using an in vitro system consisting of nuclear and nucleoid monolayers as model chromatin substrates. These substrates, derived from human skin fibroblasts, were challenged with hydroxyl radicals produced via a Fenton reaction involving Fe(II)-ethylenediaminetetraacetic acid and ascorbic acid. The resulting DNA strand breaks were measured using the alkaline unwinding technique. The sequential removal of chromosomal proteins from the DNA by pretreating nuclear monolayers with increasing concentrations of salt dramatically increased the frequency of hydroxyl radical-induced DNA strand breaks. Furthermore, the DNA in decondensed chromatin was found to contain 14-fold fewer DNA strand breaks than naked, supercoiled DNA, whereas the DNA of "native" chromatin and "condensed" chromatin contained 100-fold and 300-fold fewer breaks, respectively. We conclude that the binding of histones to the DNA and its organization into higher order chromatin structures dramatically protects the DNA against hydroxyl radical-induced DNA strand breaks and thus should be considered part of the cellular defense against the induction of oxidative DNA damage.

    View details for Web of Science ID A1992JB22100004

    View details for PubMedID 1323299

  • GENE-SPECIFIC DNA-REPAIR IN TERMINALLY DIFFERENTIATING RAT MYOBLASTS MUTATION RESEARCH Ho, L., Hanawalt, P. C. 1991; 255 (2): 123-141

    Abstract

    Preferential DNA repair in expressed genes has been well documented in proliferating mammalian cells following ultraviolet irradiation. It was of interest to learn whether excision repair is similarly selective in terminally differentiating cells. We have measured the removal of ultraviolet-induced cyclobutane pyrimidine dimers (detected as T4 endonuclease V-sensitive sites) from various genes in cultured L8 rat skeletal myoblasts. In these cells, the transcription of muscle-specific genes such as the embryonic myosin heavy chain (MHCemb) gene can be regulated by inducing cells to differentiate. L8 myoblasts are somewhat more sensitive than Chinese hamster ovary cells to ultraviolet radiation, and they exhibit relatively poor overall DNA-repair rates throughout differentiation. Irradiation severely reduces the rates of transcription and steady-state RNA levels for the genes studied. Although differences in kinetics are seen between the repair of active and inactive genes, repair rates are low relative to those previously measured in proliferating rodent cell lines. Repair efficiency in the MHCemb gene increases as it is activated during differentiation and, in fact, approaches 100% within 5 days, while that in the silent GAP43 gene is much lower. While repair efficiencies generally correlate with expression in the genes studied, the overall time course of repair appears to be prolonged in these cells compared to that in proliferating cells. These terminally differentiating cells seem to maintain a DNA damage surveillance and repair capacity for selected genes and/or genomic domains.

    View details for Web of Science ID A1991GF75400002

    View details for PubMedID 1717842

  • THE GENETIC-DEFECT IN THE CHINESE-HAMSTER OVARY CELL MUTANT UV61 PERMITS MODERATE SELECTIVE REPAIR OF CYCLOBUTANE PYRIMIDINE DIMERS IN AN EXPRESSED GENE MUTATION RESEARCH Lommel, L., Hanawalt, P. C. 1991; 255 (2): 183-191

    Abstract

    We examined removal of cyclobutane pyrimidine dimers (CPDs) from the dihydrofolate reductase (DHFR) gene in ultraviolet-irradiated Chinese hamster ovary (CHO) UV61 and UV5 cells. The sensitivity of UV61 cells to UV-irradiation is intermediate between that of the parental CHO cells and that of mutants such as UV5 that are highly defective in excision repair. UV61 cells have been characterized as having normal repair of pyrimidine(6-4)pyrimidone photoproducts (6-4 PPs) but no detectable removal of CPDs from the genome overall. We find that the extent of removal of CPDs from the DHFR gene in UV61 cells is intermediate between that of the parental CHO cells and that of the UV5 mutant, and the observed repair appears to be confined to the transcribed strand. We detected no removal of CPDs from the DHFR gene in UV5 cells. Our findings in UV61 cells demonstrate a correlation between survival after UV-irradiation and CPD repair in an expressed gene in a cell line with moderate UV-sensitivity and yet no apparent removal of CPDs from the genome as a whole. We have thus demonstrated that overall repair measurements can be misleading. Our results have implications for the determination of the relative biological importance of the CPD and the 6-4 PP, and they further support the hypothesis that removal of CPDs from transcriptionally active DNA is crucial for UV-resistance.

    View details for Web of Science ID A1991GF75400007

    View details for PubMedID 1922150

  • DIRECT EVIDENCE FOR SPATIAL AND TEMPORAL REGULATION OF TRANSFORMING GROWTH-FACTOR BETA-1 EXPRESSION DURING CUTANEOUS WOUND-HEALING JOURNAL OF CELLULAR PHYSIOLOGY Kane, C. J., Hebda, P. A., MANSBRIDGE, J. N., Hanawalt, P. C. 1991; 148 (1): 157-173

    Abstract

    The expression of transforming growth factor (TGF beta 1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGF beta 1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGF beta 1 present in normal skin as previously shown. TGF beta 1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGF beta 1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness procine skin. Anti-CC reactive TGF beta 1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGF beta 1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGF beta 1 activity. One day post-injury anti-CC reactive TGF beta 1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGF beta 1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGF beta 1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGF beta 1 activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGF beta 1 disappears while constitutive anti-LC reactive TGF beta 1 persists. Thus, changes in the structure or conformation of TGF beta 1, its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.

    View details for Web of Science ID A1991GA08100018

    View details for PubMedID 1907288

  • DIFFERENTIAL INTRODUCTION AND REPAIR OF PSORALEN PHOTOADDUCTS TO DNA IN SPECIFIC HUMAN GENES CANCER RESEARCH Islas, A. L., Vos, J. M., Hanawalt, P. C. 1991; 51 (11): 2867-2873

    Abstract

    We have developed a novel procedure to measure interstrand DNA cross-linking in specific DNA sequences. After alkaline denaturation, CsCl gradient equilibrium sedimentation at pH 10.8 is used to resolve cross-linked double-stranded DNA from un-cross-linked single-stranded DNA. The DNA in gradient fractions is slot-blotted and hybridized with 32P-labeled DNA probes for the sequences of interest. After densitometric quantitation of the autoradiograms, the fraction of DNA cross-linked is determined by the ratio of cross-linked DNA to total DNA (the sum of cross-linked and un-cross-linked DNA). We have used this approach to measure the initial levels of production and extent of repair of the photoadducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen, i.e., both interstrand cross-links and cross-linkable monoadducts, in specific DNA sequences in cultured human cells. Under conditions in which DNA fragments carrying the expressed dihydrofolate reductase gene were extensively modified, with approximately 92% of the fragments cross-linked, only 37% of the fragments containing the unexpressed fms protooncogene were cross-linked. The overall level of cross-linking for bulk DNA was 74%. Within 24 h, 90% of the cross-linking had been removed from the dihydrofolate reductase gene, whereas little removal was detected in fms, and the bulk DNA showed 31% removal. From this study, we conclude that both the introduction and removal of 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts are dependent upon the target DNA sequence and its transcriptional activity. The implications for DNA repair of chromatin structure and active transcription are discussed in relation to our results.

    View details for Web of Science ID A1991FM97800019

    View details for PubMedID 2032227

  • HETEROGENEITY OF DNA-REPAIR AT THE GENE LEVEL MUTATION RESEARCH Hanawalt, P. C. 1991; 247 (2): 203-211

    Abstract

    Overall genomic DNA repair efficiencies do not necessarily correlate with cellular sensitivities to radiation and other DNA-damaging agents. My colleagues and I have developed experimental approaches to measure DNA lesions and their repair in defined DNA sequences and we have discovered that for some types of damage, such as the cyclobutane pyrimidine dimers produced in DNA by ultraviolet light (UV), repair is highly selective for transcribed DNA strands in active genes: repair may be directly coupled to the transcription apparatus. Freely diffusing repair complexes may account for the much lower repair efficiencies observed in silent genomic domains. The viability of mammalian cells may be ensured through selective repair of transcription-blocking DNA damage in essential, expressed genes rather than as a consequence of overall genomic repair. Persisting damage in non-transcribed domains may account for some cell-specific mutagenic and carcinogenic phenomena. In UV-irradiated cells from xeroderma pigmentosum (complementation group C) there is a deficiency in the removal of pyrimidine dimers from silent genomic domains, while in Cockayne's syndrome the defect appears to involve the preferential repair of active genes. In contrast to the cancer-prone characteristic of xeroderma pigmentosum the victims of Cockayne's syndrome do not suffer enhanced skin cancer induction by sunlight. Susceptibility to cancer and other biological endpoints is clearly dependent upon the fine structure detail of the DNA repair response.

    View details for Web of Science ID A1991FH26300005

    View details for PubMedID 2011138

  • TRANSFORMING GROWTH FACTOR-BETA-1 LOCALIZATION IN NORMAL AND PSORIATIC EPIDERMAL-KERATINOCYTES INSITU JOURNAL OF CELLULAR PHYSIOLOGY Kane, C. J., KNAPP, A. M., MANSBRIDGE, J. N., Hanawalt, P. C. 1990; 144 (1): 144-150

    Abstract

    Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.

    View details for Web of Science ID A1990DN56900018

    View details for PubMedID 1694857

  • Selective DNA repair in expressed genes in mammalian cells. Progress in clinical and biological research Hanawalt, P. C. 1990; 340A: 213-222

    View details for PubMedID 2201974

  • INDUCTION OF THE ESCHERICHIA-COLI LACTOSE OPERON SELECTIVELY INCREASES REPAIR OF ITS TRANSCRIBED DNA STRAND NATURE Mellon, I., Hanawalt, P. C. 1989; 342 (6245): 95-98

    Abstract

    Nucleotide excision repair helps to ameliorate the lethal and mutagenic consequences of DNA damage by removing helix-distorting lesions from cellular genomes. We have previously analysed the removal of ultraviolet-induced cyclobutane pyrimidine dimers from specific DNA sequences in mammalian cells and demonstrated that transcriptionally active genes are preferentially repaired. Additionally, we found that in rodent and human cells only the transcribed strand of the dihydrofolate reductase gene is selectively repaired. Transcription is blocked by pyrimidine dimers in template DNA and the selective removal of these lesions seems to be important for cell survival after irradiation with ultraviolet light. To determine whether this feature of repair is common to prokaryotes and eukaryotes and better to understand its mechanism, we have investigated repair in the two separate DNA strands of the lactose operon of ultraviolet-irradiated Escherichia coli. We find a dramatic difference in the repair of the two strands only when transcription is induced. Most dimers are removed from the transcribed strand of the induced operon within five minutes of irradiation. In the nontranscribed strand, repair is significantly slower and resembles that found in both strands of the uninduced operon. Thus there seems to be a mechanism that couples nucleotide excision repair and transcription.

    View details for Web of Science ID A1989AX86800066

    View details for PubMedID 2554145

  • DNA INTERSTRAND CROSS-LINKS PROMOTE CHROMOSOMAL INTEGRATION OF A SELECTED GENE IN HUMAN-CELLS MOLECULAR AND CELLULAR BIOLOGY Vos, J. M., Hanawalt, P. C. 1989; 9 (7): 2897-2905

    Abstract

    We have used integrative pSV2 plasmids to learn how DNA lesions affect nonhomologous recombination with human chromosomes. Enhanced stable transformation of fibrosarcoma cells with a selectable gene was observed after chemical modification of the plasmid DNA; thus, cells transfected with plasmid pSV2-gpt carrying photoadducts of the cross-linking agent 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) yielded four- to sevenfold-higher levels of Gpt+ transformants than were obtained with untreated plasmid. The enhancement due to HMT interstrand cross-links was at least as great as that due to the monoadducts. DNA hybridization analysis indicated that the enhanced transformation frequency resulted from an increased number of cells carrying integrated plasmid sequences rather than from a higher copy number per transformant. The enhancement was not seen with a plasmid missing the sequences flanking the minimal simian virus 40 gpt transcription unit. Cotransfection with untreated and HMT-treated plasmids suggested that the HMT-containing DNA interacted preferentially with some cellular factor that promoted chromosomal integration of the plasmid DNA. It is concluded that (i) interstrand cross-linking as well as intrastrand DNA adducts promote nonhomologous recombination in human chromatin and (ii) DNA sequences flanking the selectable genes are the targets for such recombinational events.

    View details for Web of Science ID A1989AC45000015

    View details for PubMedID 2779552

  • REPAIR ANALYSIS OF MITOMYCIN C-INDUCED DNA CROSSLINKING IN RIBOSOMAL-RNA GENES IN LYMPHOBLASTOID-CELLS FROM FANCONIS ANEMIA PATIENTS MUTATION RESEARCH Matsumoto, A., Vos, J. M., Hanawalt, P. C. 1989; 217 (3): 185-192

    Abstract

    The repair of mitomycin C (MMC)-induced DNA crosslinking was analyzed by denaturation-renaturation gel electrophoresis in ribosomal RNA genes in lymphoblastoid cell lines from 4 patients with Fanconi's anemia (FA). In comparison to normal lymphoblastoid cell lines, 2 lines of FA cells belonging to complementation group A clearly exhibited higher sensitivity to MMC and an almost identical deficiency in the removal of DNA crosslinking. A complementation group B cell line, HSC 62, exhibited a lower sensitivity than group A cells and a lesser deficiency in crosslink repair. Another 'non-A' group cell line, HSC 230, reproducibly exhibited even higher sensitivity to MMC than group A cells. The results on MMC crosslinkage removal at the molecular level correlated well with cell survival. The observed subtle differences of repair among the 4 FA cell lines might represent possible genetic differences in the respective FA complementation groups.

    View details for Web of Science ID A1989U510800002

    View details for PubMedID 2497343

  • REPAIR OF N-METHYLPURINES IN SPECIFIC DNA-SEQUENCES IN CHINESE-HAMSTER OVARY CELLS - ABSENCE OF STRAND SPECIFICITY IN THE DIHYDROFOLATE-REDUCTASE GENE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Scicchitano, D. A., Hanawalt, P. C. 1989; 86 (9): 3050-3054

    Abstract

    We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the genome. Chinese hamster ovary cells were exposed to dimethyl sulfate, and the isolated DNA was treated with an appropriate restriction endonuclease. The DNA was heated to convert remaining N-methylpurines to apurinic sites to render them alkaline-labile. Duplicate samples heated in the presence of methoxyamine to protect the apurinic sites from alkaline hydrolysis provided controls to assess total DNA. After alkaline hydrolysis, agarose gel electrophoresis, Southern transfer, and probing for the fragment of interest, the ratios of band intensities of the test DNA sample to its methoxyamine-treated control counterpart were calculated to yield the percentage of fragments containing no alkaline-labile sites. The frequency of N-methylpurines was measured at different times after dimethyl sulfate treatment to study repair. We found no differences between the rates of repair of N-methylpurines in the active dihydrofolate reductase gene and a nontranscribed region located downstream from it in treated cells. Also, similar rates of repair were observed in the transcribed and nontranscribed strands of the gene, in contrast to previous results for the removal of cyclobutane pyrimidine dimers. Thus, there does not appear to be a coupling of N-methylpurine repair to transcription in Chinese hamster ovary cells. However, the repair in the dihydrofolate reductase domain appears to be somewhat more efficient than that in the genome overall. Our method permits the quantifying at the defined gene level of abasic sites or of any DNA adduct that can be converted to them.

    View details for Web of Science ID A1989U446200014

    View details for PubMedID 2785688

  • DEMETHYLATION ENHANCES REMOVAL OF PYRIMIDINE DIMERS FROM THE OVERALL GENOME AND FROM SPECIFIC DNA-SEQUENCES IN CHINESE-HAMSTER OVARY CELLS MOLECULAR AND CELLULAR BIOLOGY Ho, L., Bohr, V. A., Hanawalt, P. C. 1989; 9 (4): 1594-1603

    Abstract

    We have examined the effects of changes in cytosine methylation on DNA repair in UV-irradiated Chinese hamster ovary (CHO) cells. A hypomethylated derivative of the CHO K1B11 line, B11aza, was established by passaging B11 cells over several months in increasing concentrations of 5-azacytidine; greater than 60% demethylation was consistently demonstrated in these conditioned cells. Following a UV dose of 10 J/m2, the amount of repair replication performed within 24 h was approximately twofold higher in B11aza cells than in control B11 cells. Removal of T4 endonuclease V-sensitive sites (ESS) from specific restriction fragments within and around the dihydrofolate reductase (DHFR) gene was then examined in B11aza cells and compared with that in B11 cells. Although demethylation had little or no effect on repair in the 5' half of the DHFR gene, within a nontranscribed sequence immediately downstream from the gene, or within an extragenic region further downstream from the DHFR gene, significant increases in repair were observed at the 3' end of the DHFR gene and within an extragenic region upstream of the DHFR gene. However, the increases in DNA repair were not accompanied by any changes in overall cellular resistance to UV when colony-forming ability was assayed. We suggest that the level of DNA methylation may play an indirect role in the regulation of DNA repair, perhaps through an effect on chromatin structure or transcriptional activity.

    View details for Web of Science ID A1989T862900023

    View details for PubMedID 2725518

  • EFFECT OF DNA DAMAGE ON STABLE TRANSFORMATION OF MAMMALIAN-CELLS WITH INTEGRATIVE AND EPISOMAL PLASMIDS MUTATION RESEARCH Vos, J. M., Hanawalt, P. C. 1989; 220 (2-3): 205-220

    Abstract

    The efficiency of stable transformation of human cells by integrative (non-replicating) plasmids carrying a selectable gene has been shown to be markedly enhanced by the introduction into the plasmid DNA of bulky damage, such as cyclobutane pyrimidine dimers or psoralen photoadducts. Enhanced transformation (ET) occurs in all human cells tested, including DNA repair-deficient cells from the hereditary syndrome xeroderma pigmentosum, but significantly less, if at all, in rodent cells. ET has been observed with a variety of integrative plasmid constructs, suggesting the generality of the phenomenon; as expected, ET is due to an increase in the number of cells carrying integrated plasmid sequences. In contrast to integrative plasmids, stable transformation by episomal (autonomously replicating) plasmids derived from the Epstein-Barr virus is only depressed by the introduction of photoproducts; furthermore, pronounced inactivation of transformation mediated by episomal plasmids becomes apparent in xeroderma pigmentosum cells. Altogether, these results suggest that DNA damage increases the probability of stable insertion of heterologous non-replicating DNA into human chromosomes. Moreover, the differential sensitivity to DNA damage of human cell transformation mediated by integrative versus episomal plasmids suggests caution in using such assay to measure host cell reactivation capacity; processing of DNA damage in mammalian cells might differ significantly between intra- versus extra-chromosomal DNA. Since ET may be induced by damage outside the selectable gene carried on integrative plasmids, we propose a model that involves local disruption of chromatin structure by helix-distorting DNA lesions flanking actively transcribed sequences; alternatively, reorganization of such altered DNA structure might be favored by the presence of topoisomerase-like activities in the proximity of active genes. Because ET can also be induced by DNA damage to the recipient cells, it is speculated that similar mechanism(s) might be involved in the generation of other types of non-homologous DNA recombination in damaged human chromosomes, including oncogenic cell transformation mediated by integrative DNA viruses.

    View details for Web of Science ID A1989T814800018

    View details for PubMedID 2927424

  • CONCEPTS AND MODELS FOR DNA-REPAIR - FROM ESCHERICHIA-COLI TO MAMMALIAN-CELLS ENVIRONMENTAL AND MOLECULAR MUTAGENESIS Hanawalt, P. C. 1989; 14: 90-98

    Abstract

    Much of our early understanding of the mechanisms of excision-repair and its roles in maintaining genome integrity and cellular viability was derived from studies with bacteria. In fact, the discoveries of damage excision and repair replication were made in ultraviolet (UV)-irradiated Escherichia coli. Recent advances in recombinant DNA technology have helped to further our understanding of the manner in which mammalian cells deal with damage in their complex genomes. These include the discovery that expressed genes may be preferentially repaired and, furthermore, that the transcribed DNA strand, for some types of damage, is selectively repaired within an active gene. The latter finding has now been documented in E. coli as well, so it is likely that it is of widespread importance as a mechanism to ensure the expression of active genes in otherwise damaged cells. It is certain that studies with bacterial systems as models will continue to have an important impact on the development of the field of mammalian DNA repair.

    View details for Web of Science ID A1989AE31300016

    View details for PubMedID 2659338

  • DNA DAMAGE STIMULATES HUMAN CELL-TRANSFORMATION BY INTEGRATIVE BUT NOT EPISOMAL EPSTEIN-BARR VIRUS-DERIVED PLASMID MOLECULAR CARCINOGENESIS Vos, J. M., WAUTHIER, E. L., Hanawalt, P. C. 1989; 2 (5): 237-244

    Abstract

    Previous work has demonstrated that ultraviolet (UV) irradiation of SV40-based plasmids can strikingly enhance the frequency of stable transformation of human cells. In this study we compared the effect of UV-induced DNA damage on transformation mediated by integrative versus autonomously replicating plasmids derived from human Epstein-Barr virus (EBV). We report that transfection of human fibroblasts with UV-irradiated integrative EBV-based plasmid results in enhanced transformation. However, transfection of UV-damaged episomal EBV-based constructs into the same human cell line does not enhance transformation; in fact, the extrachromosomal status of the plasmid is maintained irrespective of the UV dose to the plasmid. We conclude that enhanced transformation of human cells by damaged DNA requires its chromosomal integration.

    View details for Web of Science ID A1989CF69500002

    View details for PubMedID 2557855

  • TEMPERATURE-DEPENDENT SURVIVAL OF UV-IRRADIATED ESCHERICHIA-COLI-K12 MOLECULAR & GENERAL GENETICS Ganesan, A. K., Hunt, J., Hanawalt, P. C. 1988; 214 (2): 198-203

    Abstract

    We have found that several excision deficient derivatives of Escherichia coli K12 survive better after UV irradiation if incubated at 42 degrees C than if incubated at 30 degrees C. The highest survival was observed when incubation at 42 degrees C followed UV irradiation and was maintained for at least 16 h. Our results indicate that this temperature dependent resistance (TDR) requires a functional recA gene, but not uvrA, uvrB, recF, or recB genes, or the recA441 (tif-1) mutation which allows thermoinduction of the recA-lexA regulon. Our data are consistent with the idea that the increase in survival observed at 42 degrees C reflects enhanced daughter-strand gap repair by DNA strand exchange. Although the conditions used to elicit TDR can induce heat shock proteins and thermotolerance in E. coli, the relationship between the two responses remains to be elucidated.

    View details for Web of Science ID A1988Q513500003

    View details for PubMedID 3070347

  • COMPARATIVE REMOVAL OF PYRIMIDINE DIMERS FROM HUMAN EPIDERMAL-KERATINOCYTES INVIVO AND INVITRO JOURNAL OF INVESTIGATIVE DERMATOLOGY REUSCH, M. K., Meager, K., Leadon, S. A., Hanawalt, P. C. 1988; 91 (4): 349-352

    Abstract

    We have compared the kinetics for repair of UV-induced cyclobutane pyrimidine dimers in the DNA of keratinocytes in human skin and in cell culture. A small area of the buttocks of volunteers was exposed to UVB-irradiation and biopsies were taken at various time intervals. Epidermal keratinocytes in culture from the same subjects were exposed to UVC with doses chosen to elicit comparable yields of dimers in cellular DNA. The initial density of pyrimidine dimers and the kinetics of their removal were assessed utilizing the dimer-specific T4 endonuclease V and sedimentation of the unlabeled DNA through alkaline sucrose gradients. The position of DNA in the gradients was determined using a monoclonal antibody against random sequences of single-stranded DNA in a sensitive immunoassay. The initial dimer frequency was 3.9-6.7 per 10(8) daltons DNA. About 40% of the dimers were removed within 1 h, 70% in 6 h, and 90% in 24 h for both in vivo and in vitro samples. The early rapid removal phase may represent preferential repair of actively transcribed genes. Our findings reaffirm the usefulness and applicability of cell culture systems to model in vivo repair phenomena. The use of monoclonal antibodies to detect single-stranded DNA in alkaline sucrose gradients may be of value in a variety of studies involving human tissues in which it is not possible to use radioactive labeling of the DNA for the analysis.

    View details for Web of Science ID A1988Q217500014

    View details for PubMedID 2459263

  • HIGH-EFFICIENCY TRANSFORMATION OF BACTERIAL-CELLS BY ELECTROPORATION JOURNAL OF BACTERIOLOGY CALVIN, N. M., Hanawalt, P. C. 1988; 170 (6): 2796-2801

    Abstract

    We have developed a method for efficiently generating transient pores in the outer membranes of Escherichia coli K-12 derivatives by using a new type of electroporation apparatus. The pores are large enough and persist long enough to facilitate the equilibration of plasmid molecules between the intracellular and extracellular spaces. The method has been used to transform bacterial cells with an efficiency greater than 10(9) transformants per microgram of plasmid. It has also been used to extract intact plasmid from transformed cells with efficiencies comparable to those of the traditional alkaline lysis or CsCl equilibrium density gradient techniques. The technique is simple and rapid, allowing a transformation or the preparation of microgram quantities of plasmid to be accomplished in minutes.

    View details for Web of Science ID A1988N699900056

    View details for PubMedID 3286620

  • ROLE OF TRANSFORMING GROWTH FACTOR-BETA IN THE MATURATION OF HUMAN EPIDERMAL-KERATINOCYTES JOURNAL OF INVESTIGATIVE DERMATOLOGY MANSBRIDGE, J. N., Hanawalt, P. C. 1988; 90 (3): 336-341

    Abstract

    Changes in protein synthesis and phosphorylation in cultured human keratinocytes in response to TGF-beta have been examined by one and two dimensional electrophoresis. Transforming growth factor beta has been shown to cause little change in the rate of methionine incorporation in the concentration range in which growth is reversibly arrested. It does, however, prevent the labeling of certain specific bands detected on gels of triton-soluble proteins after 3 days of treatment. Phosphorylation of triton-soluble proteins is inhibited at concentrations of TGF-beta rather higher than the Kd of its receptor and may represent a nonphysiological effect. Nonetheless, the phosphorylation of certain prominent species is reduced. In keratinocytes cultured in delipidated serum, which show some expression of keratin 1 (67 kd) characteristic of normal maturation, TGF-beta reduces the incorporation of methionine into this keratin 1 and increases labeling of keratins 6 and 16. Transforming growth factor beta thus promotes regenerative maturation, which is normally expressed during wound healing. The ability of TGF-beta to arrest keratinocyte growth in a reversible manner and to stimulate regenerative maturation, supports its physiological role in controlling the balance between cell division, migration and maturation during epidermal wound healing.

    View details for Web of Science ID A1988M543400015

    View details for PubMedID 2450142

  • ENHANCED TRANSFORMING ACTIVITY OF PSV2 PLASMIDS IN HUMAN-CELLS DEPENDS UPON THE TYPE OF DAMAGE INTRODUCED INTO THE PLASMID MUTATION RESEARCH Spivak, G., Leadon, S. A., Vos, J. M., Meade, S., Hanawalt, P. C., Ganesan, A. K. 1988; 193 (2): 97-108

    Abstract

    When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.

    View details for Web of Science ID A1988M516700002

    View details for PubMedID 2831452

  • PREFERENTIAL DNA-REPAIR IN EXPRESSED GENES ENVIRONMENTAL HEALTH PERSPECTIVES Hanawalt, P. C. 1987; 76: 9-14

    Abstract

    Potentially deleterious alterations to DNA occur nonrandomly within the mammalian genome. These alterations include the adducts produced by many chemical carcinogens, but not the UV-induced cyclobutane pyrimidine dimer, which may be an exception. Recent studies in our laboratory have shown that the excision repair of pyrimidine dimers and certain other lesions is nonrandom in the mammalian genome, exhibiting a distinct preference for actively transcribed DNA sequences. An important consequence of this fact is that mutagenesis and carcinogenesis may be determined in part by the activities of the relevant genes. Repair may also be processive, and a model is proposed in which excision repair is coupled to transcription at the nuclear matrix. Similar but freely diffusing repair complexes may account for the lower overall repair efficiencies in the silent domains of the genome. Risk assessment in relation to chemical carcinogenesis requires assays that determine effective levels of DNA damage for producing malignancy. The existence of nonrandom repair in the genome casts into doubt the reliability of overall indicators of DNA binding and lesion repair for such determinations. Furthermore, some apparent differences between the intragenomic repair heterogeneity in rodent cells and that in human cells mandate a reevaluation of rodent test systems for human risk assessment. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response.

    View details for Web of Science ID A1987M423800002

    View details for PubMedID 3447906

  • SELECTIVE REMOVAL OF TRANSCRIPTION-BLOCKING DNA DAMAGE FROM THE TRANSCRIBED STRAND OF THE MAMMALIAN DHFR GENE CELL Mellon, I., Spivak, G., Hanawalt, P. C. 1987; 51 (2): 241-249

    Abstract

    We find a dramatic difference in the efficiency of removal of UV-induced pyrimidine dimers from the transcribed and nontranscribed strands of the dihydrofolate reductase (DHFR) gene in cultured hamster and human cells. In hamster cells, 80% of the dimers are removed from the transcribed strand in 4 hr, but little repair occurs in the nontranscribed strand even after 24 hr. In human cells, repair is significantly faster in the transcribed strand than in the other strand. Furthermore, in the 5' flanking region of the human DHFR gene, selective rapid repair occurs in the opposite DNA strand relative to the transcribed strand of the DHFR gene. This strand is thought to serve as a template for transcription of a divergent transcript. These results have important implications for excision repair pathways and mutagenesis in mammalian cells.

    View details for Web of Science ID A1987K595600009

    View details for PubMedID 3664636

  • ENHANCED TRANSFORMING ACTIVITY OF ULTRAVIOLET-IRRADIATED PSV2-GPT IS DUE TO DAMAGE OUTSIDE THE GPT TRANSCRIPTION UNIT PLASMID Leadon, S. A., Ganesan, A. K., Hanawalt, P. C. 1987; 18 (2): 135-141

    Abstract

    We have shown that when pSV2-gpt is introduced into human cells by calcium phosphate coprecipitation, the yield of Gpt+ transformants is increased by irradiating the plasmid with 254 nm uv. To elucidate the mechanism underlying this response, we constructed pSV2-gpt molecules in which the uv damage was confined to a particular region: a 3.0-kb region containing the pBR322 sequences and simian virus 40 (SV40) sequences not required for expression of the gpt gene, or a 2.3-kb fragment containing the Escherichia coli gpt gene together with the SV40 early promoter and sequences needed for splicing and polyadenylation. The transforming activity of the plasmid was greatly enhanced by uv damage confined to the 3.0-kb pBR322 region and increased linearly with uv dose up to 1 kJ/m2, but remained relatively constant at doses between 2 and 8 kJ/m2. Positioning the damaged region upstream, or both upstream and downstream, from the gpt transcription unit increased the uv enhancement slightly compared to positioning the damaged region only downstream. In contrast, transforming activity was significantly decreased by damage in the 2.3-kb gpt transcription unit. These results suggest that uv damage outside a selectable marker gene in a plasmid can increase the probability of stable integration of the plasmid into the genome of recipient cells without inhibiting expression of of the gene.

    View details for Web of Science ID A1987L108600005

    View details for PubMedID 2829251

Conference Proceedings


  • Role of transcription-coupled DNA repair in susceptibility to environmental carcinogenesis Hanawalt, P. C. US DEPT HEALTH HUMAN SCIENCES PUBLIC HEALTH SCIENCE. 1996: 547-551

    Abstract

    Susceptibility to environmental carcinogenesis is the consequence of a complex interplay between intrinsic hereditary factors and actual exposures to potential carcinogenic agents. We must learn the nature of these interactions as well as the genetic defects that confer enhanced risk. In some genetic diseases an increased cancer risk correlates with a defect in the repair or replications of damaged DNA. Examples include xeroderma pigmentosum (XP), ataxia telangiectasia, Fanconi's anemia, and Bloom's syndrome. In Cockayne's syndrome the Specific defect in transcription-coupled repair (TCR) does not predispose the patients to the sunlight-induced skin cancer characteristic of XP. The demonstration of TCR in the XP129 partial revertant of XP-A cells indicates that ultraviolet (UV) resistance correlates with repair of cyclobutane pyrimidine dimers in active genes. Repair measured as an average over the genome can be misleading, and it is necessary to consider genomic locations of DNA damage and repair for a meaningful assessment of the biological importance of particular DNA lesions. Mutations in the p53 tumor suppressor gene are found in many human tumors. TCR accounts for the resulting mutational spectra in the p53 gene in certain tumors. Li-Fraumeni syndrome fibroblasts expressing only mutant p53 are more UV-resistant and exhibit less UV-induced apoptosis than normal human cells or heterozygotes for mutations in only one allele of p53. The p53-defective cells are deficient in global excision repair capacity but have retained TCR. The loss of p53 function may lead to greater genomic instability by reducing the efficiency of global DNA repair while cellular resistance may be assured through the operation of TCR and the elimination of apoptosis.

    View details for Web of Science ID A1996UQ08400027

    View details for PubMedID 8781381

  • Fine structure mapping of DNA repair within a 100 kb genomic region in Chinese hamster ovary cells Spivak, G., Hanawalt, P. C. ELSEVIER SCIENCE BV. 1996: 207-216

    Abstract

    We have investigated at a high level of resolution the repair of cyclobutane pyrimidine dimers (CPD) in a large amplified genomic region in Chinese hamster ovary B11 cells. We found strand selective repair in DNA fragments within two active genes, DHFR and an unknown gene adjacent to DHFR. These genes generate divergent transcripts from the same promoter region; their transcribed strands were virtually free of CPD within 24 h after irradiation with 10 j/m2 of ultraviolet light (254nm), while their non-transcribed strands were poorly repaired. We also examined the repair of CPD in three DNA fragments within a 50 kb region downstream of DHFR, in which two origins of replication flanking a matrix attachment site have been characterized from independently derived cell lines with amplified DHFR domains; repair of CPD in this non-transcribed region was similarly poor in both DNA strands. Transcription-coupled repair of CPD in the DHFR gene exhibited the same proficiency throughout the transcription unit: analysis of the efficiency of removal of CPD over time revealed no differences between repair in the 5' and the 3' ends of the DHFR gene. Implications for mechanisms of transcription-coupled repair are discussed.

    View details for Web of Science ID A1996TY15000024

    View details for PubMedID 8657183

  • GENOMIC HETEROGENEITY OF DNA-REPAIR - ROLE IN AGING Hanawalt, P. C., Gee, P., Ho, L., Hsu, R. K., Kane, C. J. NEW YORK ACAD SCIENCES. 1992: 17-25

    Abstract

    The introduction and repair of DNA lesions are generally heterogeneous with respect to different genomic domains. In particular, the repair of helix-distorting damage, such as the cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light occurs selectively in expressed genes. This is due in large part to the preferential repair of transcribed DNA strands, which is then reflected in a bias toward mutagenesis from persisting lesions in nontranscribed strands. Consequently, determination of overall genomic repair efficiencies may not be a good indicator of cellular sensitivity to agents that damage DNA. Although some studies suggest an age-related accumulation of altered nucleotides in DNA, we do not know the intragenomic distribution of those changes and whether they are relevant to the physiological aspects of aging. Subtle changes in the pattern of preferential repair during maturation could have profound effects on cell and tissue function. DNA repair has been analyzed in differentiating cell systems as possible models for aging. We have observed attenuated overall repair of CPD in differentiated rat myoblasts or PC12 neuron-like cells. In both model systems, several expressed genes have been shown to be repaired relatively efficiently but without strand specificity. In another model system of human HT1080 fibroblasts differentiating in the presence of dexamethasone, we demonstrated enhanced repair in the gene for plasminogen activator inhibitor I whose transcription is induced and, correspondingly, a reduced repair rate in the urokinase plasminogen activator gene whose transcription is suppressed. We conclude that any attempted correlation of the phenomena of aging with DNA repair should focus on the relevant genes in the tissue of interest.

    View details for Web of Science ID A1992KJ58100003

    View details for PubMedID 1482051

  • SELECTIVE DNA-REPAIR IN ACTIVE GENES Hanawalt, P. C. AKADEMIAI KIADO RT. 1990: 77-91

    Abstract

    My colleagues and I have discovered intragenomic heterogeneity in DNA repair in mammalian cells. Consequences of unrepaired DNA damage depend upon the precise location of the damage with respect to relevant genes. It is therefore important to understand rules governing accessibility of specific DNA sequences in chromatin to damage and repair. The efficiency of removal of pyrimidine dimers has been mapped in the active dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. Repair within the gene was shown to be much more efficient than that in silent downstream sequences or in the genome overall. Preferential repair of active and essential genes such as DHFR may account for the fact that rodent cells are as UV-resistant as human cells in spite of their much lower overall repair efficiencies. In repair proficient human cells the rate of repair in the DHFR gene is greater than that in the overall genome or in non-transcribed alpha DNA sequences. The efficiency of removal of pyrimidine dimers is much higher in the transcribed than the non-transcribed DNA strands of the DHFR gene in both CHO and human cells. An excision-repair complex may be directly coupled to the transcription machinery to ensure early removal of transcription-blocking lesions in active genes. Sequences in the active c-abl protooncogene are repaired much more efficiently than are sequences containing the inactive c-mos protooncogene in Swiss mouse 3T3 cells. Tissue specific and cell specific differences in the coordinate regulation of protooncogene expression and DNA repair may account for corresponding differences in the carcinogenic response. Efficient replicative bypass of persisting psoralen monoadducts, but not interstrand crosslinks, was demonstrated in the human DHFR gene. It is likely that most bulky lesions in mammalina DNA, other than crosslinks, do not pose insurmountable problems for replication in vivo, but they must be removed from essential transcribed sequences to maintain cellular viability.

    View details for Web of Science ID A1990FA42900009

    View details for PubMedID 2094132

  • PREFERENTIAL REPAIR OF DAMAGE IN ACTIVELY TRANSCRIBED DNA-SEQUENCES INVIVO Hanawalt, P. C. NATL RESEARCH COUNCIL CANADA. 1989: 605-611

    Abstract

    My colleagues and I have discovered intragenomic heterogeneity in DNA repair in mammalian cells. Consequences of unrepaired DNA damage depend upon the precise location of the damage with respect to relevant genes. It is therefore important to understand rules governing accessibility of specific DNA sequences in chromatin to damage and repair. The efficiency of removal of pyrimidine dimers has been determined in the active dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. Repair within the gene was shown to be much more efficient than that in nontranscribed downstream sequences or in the genome overall. Preferential repair of active and essential genes such as DHFR may account for the fact that rodent cells are as uv-resistant as human cells in spite of their much lower overall repair efficiencies. In repair-proficient human cells the rate of repair in the DHFR gene is greater than that in the overall genome or in nontranscribed alpha-DNA sequences. The efficiency of removal of pyrimidine dimers is much higher in the transcribed than the nontranscribed DNA strands of the DHFR gene in both CHO and human cells. An excision-repair complex may be directly coupled to the transcription machinery to ensure early removal of transcription-blocking lesions in active genes. Sequences in the active c-abl proto-oncogene are repaired much more efficiently than are sequences containing the inactive c-mos proto-oncogene in Swiss mouse 3T3 cells. Tissue-specific and cell-specific differences in the coordinate regulation of proto-oncogene expression and DNA repair may account for corresponding differences in the carcinogenic response.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1989CL22100018

    View details for PubMedID 2698835

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