Bio

Clinical Focus


  • Cancer > GI Oncology
  • Oncology

Academic Appointments


Honors & Awards


  • Giulio Racah Prize, International School of Subnuclear Physics, Erice, Italy (1973)
  • Rita Allen Award, Rita Allen Foundation (1988-1993)
  • Clinical Scientist Award for Translational Research, Burroughs-Wellcome Fund (1997-2002)
  • Kaiser Award for Preclinical Teaching, Stanford University (2003, 2007)

Professional Education


  • Fellowship:Stanford University School of Medicine (1986) CA
  • Residency:Massachusetts General Hospital (1982) MA
  • M.D., Harvard, Medicine (1980)
  • Ph.D., M.I.T., Physics (1973)
  • A.B., Princeton, Physics (1967)
  • Board Certification: Internal Medicine, American Board of Internal Medicine (1983)
  • Medical Education:Harvard Medical School (1980) MA

Research & Scholarship

Current Research and Scholarly Interests


Our laboratory focuses on understanding how cells respond to DNA damage. Our research currently involves three areas that interact with each other: repair of ionizing radiation damage, repair of ultraviolet radiation damage, and transcriptional responses to DNA damage in cancer patients.

To understand how cells target DNA damaged by ultraviolet radiation for nucleotide excision repair, we identified UV-damaged DNA binding activity (UV-DDB). We showed that the p48 gene, encoding one of the subunits of UV-DDB, is mutated in xeroderma pigmentosum group E patients, and transcriptionally activated after DNA damage by p53. UV-DDB enhances global genomic repair and suppresses UV-induced mutagenesis.

To understand how cells repair DNA damaged by ionizing radiation, we study the non-homologous end joining pathway, which repairs double-strand breaks produced by ionizing radiation and V(D)J recombination. We wish to understand how joining optimizes the preservation of DNA sequence. We are currently studying the joining reaction with purified proteins to determine how the DNA ends are brought together, processed, and rejoined.

We have used microarrays to study the transcriptional response to ionizing radiation in lymphoblastoid cells from cancer patients with adverse reactions to radiation therapy. To interpret the microarray data, we invented new methods to analyze microarray data, including SAM (Significance Analysis of Microarrays). We successfully identified genes whose transcriptional responses predict risk for radiation toxicity. These results provide hope that treatment toxicity will soon be predicted by clinical tests.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • Cooperative Assembly of a Protein-DNA Filament for Nonhomologous End Joining JOURNAL OF BIOLOGICAL CHEMISTRY Tsai, C. J., Chu, G. 2013; 288 (25): 18110-18120

    Abstract

    Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL) and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3' overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, while XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to 5 molecules each of XL and XLF into a Ku:XL:XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to 6 molecules each of Ku and XL into a Ku:XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular weight, MEnd ligase-DNA had a lower electrophoretic mobility than Ku:XL-DNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggest that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation.

    View details for DOI 10.1074/jbc.M113.464115

    View details for Web of Science ID 000320721900017

    View details for PubMedID 23620595

  • Local false discovery rate facilitates comparison of different microarray experiments NUCLEIC ACIDS RESEARCH Hong, W., Tibshirani, R., Chu, G. 2009; 37 (22): 7483-7497

    Abstract

    The local false discovery rate (LFDR) estimates the probability of falsely identifying specific genes with changes in expression. In computer simulations, LFDR <10% successfully identified genes with changes in expression, while LFDR >90% identified genes without changes. We used LFDR to compare different microarray experiments quantitatively: (i) Venn diagrams of genes with and without changes in expression, (ii) scatter plots of the genes, (iii) correlation coefficients in the scatter plots and (iv) distributions of gene function. To illustrate, we compared three methods for pre-processing microarray data. Correlations between methods were high (r = 0.84-0.92). However, responses were often different in magnitude, and sometimes discordant, even though the methods used the same raw data. LFDR complements functional assessments like gene set enrichment analysis. To illustrate, we compared responses to ultraviolet radiation (UV), ionizing radiation (IR) and tobacco smoke. Compared to unresponsive genes, genes responsive to both UV and IR were enriched for cell cycle, mitosis, and DNA repair functions. Genes responsive to UV but not IR were depleted for cell adhesion functions. Genes responsive to tobacco smoke were enriched for detoxification functions. Thus, LFDR reveals differences and similarities among experiments.

    View details for DOI 10.1093/nar/gkp813

    View details for Web of Science ID 000272935000021

    View details for PubMedID 19825981

  • Here Comes the Sun: Recognition of UV-Damaged DNA CELL Chu, G., Yang, W. 2008; 135 (7): 1172-1174

    Abstract

    The first step in the repair of DNA damage is lesion detection. In this issue, Scrima et al. (2008) report the structure of the complex of DNA Damage-Binding Protein 1 (DDB1) and DDB2 bound to a DNA photodimer, providing critical insight into the repair of DNA damage caused by ultraviolet light.

    View details for DOI 10.1016/j.cell.2008.12.015

    View details for Web of Science ID 000261989800009

    View details for PubMedID 19109889

  • Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tsai, C. J., Kim, S. A., Chu, G. 2007; 104 (19): 7851-7856

    Abstract

    Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation or V(D)J recombination of the immunoglobulin genes. The breaks often leave mismatched or nonligatable ends, and NHEJ must repair the breaks with high efficiency and minimal nucleotide loss. Here, the NHEJ proteins Ku, DNA-dependent protein kinase catalytic subunit, XRCC4/Ligase IV, and Cernunnos/XRCC4-like factor joined mismatched and noncohesive DNA ends in the absence of processing factors. Depending on the mismatch, Cernunnos stimulated joining 8- to 150-fold. For substrates with a blunt end and a 3' overhanging end, Ku, XRCC4/Ligase IV, and Cernunnos ligated the 3' overhanging hydroxyl group to the 5' phosphate of the blunt end, leaving the other strand unjoined. This activity provides a mechanism for retaining 3' overhang sequences, as observed during V(D)J recombination in vivo. Thus, Cernunnos/XRCC4-like factor promotes a mismatched end (MEnd) DNA ligase activity to facilitate joining and to preserve DNA sequence. Furthermore, MEnd ligase activity may have applications in recombinant DNA technology.

    View details for DOI 10.1073/pnas.0702620104

    View details for Web of Science ID 000246461500024

    View details for PubMedID 17470781

  • Processing of DNA for nonhomologous end-joining is controlled by kinase activity and XRCC4/Ligase IV JOURNAL OF BIOLOGICAL CHEMISTRY Budman, J., Kim, S. A., Chu, G. 2007; 282 (16): 11950-11959

    Abstract

    Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence.

    View details for DOI 10.1074/jbc.M610058200

    View details for Web of Science ID 000245941900038

    View details for PubMedID 17272270

  • Processing of DNA for nonhomologous end-joining by cell-free extract EMBO JOURNAL Budman, J., Chu, G. 2005; 24 (4): 849-860

    Abstract

    In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. We have developed a cell-free system capable of processing and joining noncompatible DNA ends. The system had key features of NHEJ in vivo, including dependence on Ku, DNA-PKcs, and XRCC4/Ligase4. The NHEJ reaction had striking properties. Processing of noncompatible ends involved polymerase and nuclease activities that often stabilized the alignment of opposing ends by base pairing. To achieve this, polymerase activity efficiently synthesized DNA across discontinuities in the template strand, and nuclease activity removed a limited number of nucleotides back to regions of microhomology. Processing was suppressed for DNA ends that could be ligated directly, biasing the reaction to preserve DNA sequence and maintain genomic integrity. DNA sequence internal to the ends influenced the spectrum of processing events for noncompatible ends. Furthermore, internal DNA sequence strongly influenced joining efficiency, even in the absence of processing. These results support a model in which DNA-PKcs plays a central role in regulating the processing of ends for NHEJ.

    View details for DOI 10.1038/sj.emboj.7600563

    View details for Web of Science ID 000227494900018

    View details for PubMedID 15692565

  • Toxicity from radiation therapy associated with abnormal transcriptional responses to DNA damage PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rieger, K. E., Hong, W. J., Tusher, V. G., Tang, J., Tibshirani, R., Chu, G. 2004; 101 (17): 6635-6640

    Abstract

    Toxicity from radiation therapy is a grave problem for cancer patients. We hypothesized that some cases of toxicity are associated with abnormal transcriptional responses to radiation. We used microarrays to measure responses to ionizing and UV radiation in lymphoblastoid cells derived from 14 patients with acute radiation toxicity. The analysis used heterogeneity-associated transformation of the data to account for a clinical outcome arising from more than one underlying cause. To compute the risk of toxicity for each patient, we applied nearest shrunken centroids, a method that identifies and cross-validates predictive genes. Transcriptional responses in 24 genes predicted radiation toxicity in 9 of 14 patients with no false positives among 43 controls (P = 2.2 x 10(-7)). The responses of these nine patients displayed significant heterogeneity. Of the five patients with toxicity and normal responses, two were treated with protocols that proved to be highly toxic. These results may enable physicians to predict toxicity and tailor treatment for individual patients.

    View details for DOI 10.1073/pnas.0307761101

    View details for Web of Science ID 000221107900056

    View details for PubMedID 15096622

  • Significance analysis of microarrays applied to the ionizing radiation response PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tusher, V. G., Tibshirani, R., Chu, G. 2001; 98 (9): 5116-5121

    Abstract

    Microarrays can measure the expression of thousands of genes to identify changes in expression between different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. We describe a method, Significance Analysis of Microarrays (SAM), that assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). When the transcriptional response of human cells to ionizing radiation was measured by microarrays, SAM identified 34 genes that changed at least 1.5-fold with an estimated FDR of 12%, compared with FDRs of 60 and 84% by using conventional methods of analysis. Of the 34 genes, 19 were involved in cell cycle regulation and 3 in apoptosis. Surprisingly, four nucleotide excision repair genes were induced, suggesting that this repair pathway for UV-damaged DNA might play a previously unrecognized role in repairing DNA damaged by ionizing radiation.

    View details for Web of Science ID 000168311500058

    View details for PubMedID 11309499

  • Electrophoretic mobility shift assays for protein-DNA complexes involved in DNA repair. Methods in molecular biology (Clifton, N.J.) Tsai, C., Smider, V., Hwang, B. J., Chu, G. 2012; 920: 53-78

    Abstract

    The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents. UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the anticancer drug cisplatin. Ku, XRCC4/Ligase IV, and DNA-PKcs, which are involved in the repair of DNA double-strand breaks by nonhomologous end joining, assemble in complexes at DNA ends. This chapter will describe several EMSA protocols for detecting different DNA repair protein-DNA complexes. To obtain additional information, one can apply variations of the EMSA, which include the reverse EMSA to detect binding of (35)S-labeled protein to damaged DNA, and the antibody supershift assay to detect the presence of a specific protein in the protein-DNA complex.

    View details for DOI 10.1007/978-1-61779-998-3_5

    View details for PubMedID 22941596

  • An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells PLOS ONE Glotzbach, J. P., Januszyk, M., Vial, I. N., Wong, V. W., Gelbard, A., Kalisky, T., Thangarajah, H., Longaker, M. T., Quake, S. R., Chu, G., Gurtner, G. C. 2011; 6 (6)

    Abstract

    An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

    View details for DOI 10.1371/journal.pone.0021211

    View details for Web of Science ID 000292033700046

    View details for PubMedID 21731674

  • Crystal structure of human XLF: A twist in nonhomologous DNA end-joining MOLECULAR CELL Andres, S. N., Modesti, M., Tsai, C. J., Chu, G., Junop, M. S. 2007; 28 (6): 1093-1101

    Abstract

    DNA double-strand breaks represent one of the most severe forms of DNA damage in mammalian cells. One pathway for repairing these breaks occurs via nonhomologous end-joining (NHEJ) and depends on XRCC4, LigaseIV, and Cernunnos, also called XLF. Although XLF stimulates XRCC4/LigaseIV to ligate mismatched and noncohesive DNA ends, the mechanistic basis for this function remains unclear. Here we report the structure of a partially functional 224 residue N-terminal fragment of human XLF. Despite only weak sequence similarity, XLF(1-170) shares structural homology with XRCC4(1-159). However, unlike the highly extended 130 A helical domain observed in XRCC4, XLF adopts a more compact, folded helical C-terminal region involving two turns and a twist, wrapping back to the structurally conserved N terminus. Mutational analysis of XLF and XRCC4 reveals a potential interaction interface, suggesting a mechanism for how XLF stimulates the ligation of mismatched ends.

    View details for DOI 10.1016/j.molcel.2007.10.024

    View details for Web of Science ID 000252170000015

    View details for PubMedID 18158905

  • Electrophoretic mobility shift assays to study protein binding to damaged DNA. Methods in molecular biology (Clifton, N.J.) Smider, V., Hwang, B. J., Chu, G. 2006; 314: 323-344

    Abstract

    The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA-PKcs, which are involved in double-strand break repair. This chapter describes EMSA protocols for detecting proteins that bind to UV-damaged DNA, cisplatin-damaged DNA, and DNA ends. The chapter also describes variations of the EMSA that can be used to obtain additional information about these important proteins. The variations include the reverse EMSA, which can detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay, which can define the composition of protein-DNA complexes.

    View details for PubMedID 16673891

  • Assays for nonhomologous end joining in extracts DNA REPAIR, PT A Budman, J., Chu, G. 2006; 408: 430-444

    Abstract

    In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double strand breaks created by ionizing radiation and V(D)J recombination. Using human whole cell extracts prepared by the method of Baumann and West (1998), we have described a cell-free system for NHEJ that joins both compatible and noncompatible DNA ends (Budman and Chu, 2005). To measure joining efficiency and assess the processing of DNA ends, we developed a quantitative polymerase chain reaction assay for the joining of two specific DNA ends. The in vitro NHEJ reaction recapitulates key features of NHEJ observed in vivo: end joining is dependent on DNA-PK and XRCC4/Ligase4, and noncompatible ends are processed by polymerase and nuclease activities that often stabilize the alignment of opposing ends by base pairing. This chapter describes methods for preparing whole cell extracts and for studying the NHEJ reaction in vitro.

    View details for DOI 10.1016/S0076-6879(06)08027-X

    View details for Web of Science ID 000238224100027

    View details for PubMedID 16793385

  • Portrait of transcriptional responses to ultraviolet and ionizing radiation in human cells NUCLEIC ACIDS RESEARCH Rieger, K. E., Chu, G. 2004; 32 (16): 4786-4803

    Abstract

    To understand the human response to DNA damage, we used microarrays to measure transcriptional responses of 10 000 genes to ionizing radiation (IR) and ultraviolet radiation (UV). To identify bona fide responses, we used cell lines from 15 individuals and a rigorous statistical method, Significance Analysis of Microarrays (SAM). By exploring how sample number affects SAM, we rendered a portrait of the human damage response with a degree of accuracy unmatched by previous studies. By showing how SAM can be used to estimate the total number of responsive genes, we discovered that 24% of all genes respond to IR and 32% respond to UV, although most responses were less than 2-fold. Many genes were involved in known damage-response pathways for cell cycling and proliferation, apoptosis, DNA repair or the stress response. However, the majority of genes were involved in unexpected pathways, with functions in signal transduction, RNA binding and editing, protein synthesis and degradation, energy metabolism, metabolism of macromolecular precursors, cell structure and adhesion, vesicle transport, or lysosomal metabolism. Although these functions were not previously associated with the damage response in mammals, many were conserved in yeast. These insights reveal new directions for studying the human response to DNA damage.

    View details for DOI 10.1093/nar/gkh783

    View details for Web of Science ID 000224207500009

    View details for PubMedID 15356296

  • Class prediction by nearest shrunken centroids, with applications to DNA microarrays STATISTICAL SCIENCE Tibshirani, R., Hastie, T., Narasimhan, B., Chu, G. 2003; 18 (1): 104-117
  • Interaction between UV-damaged DNA binding activity proteins and the c-Abl tyrosine kinase JOURNAL OF BIOLOGICAL CHEMISTRY Cong, F., Tang, J., Hwang, B. J., Vuong, B. Q., Chu, G., Goff, S. P. 2002; 277 (38): 34870-34878

    Abstract

    The c-Abl tyrosine kinase is activated by some forms of DNA damage, including ionizing radiation, but not UV radiation. The functions of this activation in the damage response pathways remain obscure. To identify potential targets of c-Abl kinase, we utilized the yeast two-hybrid system to screen a murine cDNA library. One c-Abl binding protein of particular interest was the large subunit (DDB1) of the heterodimeric complex with UV-damaged DNA binding activity (UV-DDB). This complex binds with high specificity to DNA damaged by UV, is absent in a subset of xeroderma pigmentosum group E cells, and is required for global genomic repair of UV-induced damage. The association of c-Abl with DDB1 required the kinase domain of c-Abl and preserved the interaction between DDB1 and the small subunit (DDB2) of the UV-DDB complex. Significantly, overexpression of c-Abl increased tyrosine phosphorylation of DDB2 and suppressed UV-DDB activity. Conversely, a dominant negative, kinase-deficient allele of c-Abl decreased tyrosine phosphorylation of DDB2 and dramatically stimulated UV-DDB activity. These results suggest that one role of c-Abl may be to negatively regulate UV-DDB activity by phosphorylation of DDB2.

    View details for DOI 10.1074/jbc.M204416200

    View details for Web of Science ID 000178117000029

    View details for PubMedID 12107171

  • Xeroderma pigmentosum complementation group E and UV-damaged DNA-binding protein DNA REPAIR Tang, J., Chu, G. 2002; 1 (8): 601-616

    Abstract

    UV-damaged DNA-binding protein (UV-DDB) is composed of two subunits, DDB1 (p127) and DDB2 (p48). Mutations in the DDB2 gene inactivate UV-DDB in individuals from complementation group E of xeroderma pigmentosum (XP-E), an autosomal recessive disease characterized by sun sensitivity, severe risk for skin cancer and defective nucleotide excision repair. UV-DDB is also deficient in many rodent tissues, exposing a shortcoming in rodent models for cancer. In vitro, UV-DDB binds to cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts and other DNA lesions, stimulating the excision of CPDs, and to a lesser extent, of 6-4 photoproducts. In vivo, UV-DDB plays an important role in the p53-dependent response of mammalian cells to DNA damage. When cells are exposed to UV, the resulting accumulation of p53 activates DDB2 transcription, which leads to increased levels of UV-DDB. Binding of UV-DDB to CPDs targets these lesions for global genomic repair, suppressing mutations without affecting UV survival. Apparently, cells are able to survive with unrepaired CPDs because of the activity of bypass DNA polymerases. Finally, there is evidence that UV-DDB may have roles in the cell that are distinct from DNA repair.

    View details for Web of Science ID 000178248700002

    View details for PubMedID 12509284

  • Synapsis of DNA ends by DNA-dependent protein kinase EMBO JOURNAL DeFazio, L. G., Stansel, R. M., Griffith, J. D., Chu, G. 2002; 21 (12): 3192-3200

    Abstract

    The catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) is required for a non-homologous end-joining pathway that repairs DNA double-strand breaks produced by ionizing radiation or V(D)J recombination; however, its role in this pathway has remained obscure. Using a neutravidin pull-down assay, we found that DNA-PK(CS) mediates formation of a synaptic complex containing two DNA molecules. Furthermore, kinase activity was cooperative with respect to DNA concentration, suggesting that activation of the kinase occurs only after DNA synapsis. Electron microscopy revealed complexes of two DNA ends brought together by two DNA-PK(CS) molecules. Our results suggest that DNA-PK(CS) brings DNA ends together and then undergoes activation of its kinase, presumably to regulate subsequent steps for processing and ligation of the ends.

    View details for Web of Science ID 000176587600034

    View details for PubMedID 12065431

  • Diagnosis of multiple cancer types by shrunken centroids of gene expression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tibshirani, R., Hastie, T., Narasimhan, B., Chu, G. 2002; 99 (10): 6567-6572

    Abstract

    We have devised an approach to cancer class prediction from gene expression profiling, based on an enhancement of the simple nearest prototype (centroid) classifier. We shrink the prototypes and hence obtain a classifier that is often more accurate than competing methods. Our method of "nearest shrunken centroids" identifies subsets of genes that best characterize each class. The technique is general and can be used in many other classification problems. To demonstrate its effectiveness, we show that the method was highly efficient in finding genes for classifying small round blue cell tumors and leukemias.

    View details for Web of Science ID 000175637300012

    View details for PubMedID 12011421

  • p53 binds and activates the xeroderma pigmentosum DDB2 gene in humans but not mice MOLECULAR AND CELLULAR BIOLOGY Tan, T., Chu, G. 2002; 22 (10): 3247-3254

    Abstract

    The DDB2 gene, which is mutated in xeroderma pigmentosum group E, enhances global genomic repair of cyclobutane pyrimidine dimers and suppresses UV-induced mutagenesis. Because DDB2 transcription increases after DNA damage in a p53-dependent manner, we searched for and found a region in the human DDB2 gene that binds and responds transcriptionally to p53. The corresponding region in the mouse DDB2 gene shared significant sequence identity with the human gene but was deficient for p53 binding and transcriptional activation. Furthermore, when mouse cells were exposed to UV, DDB2 transcription remained unchanged, despite the accumulation of p53 protein. These results demonstrate direct activation of the human DDB2 gene by p53. They also explain an important difference in DNA repair between humans and mice and show how mouse models can be improved to better reflect cancer susceptibility in humans.

    View details for DOI 10.1128/MCB.22.10.3247-3254.2002

    View details for Web of Science ID 000175323900004

    View details for PubMedID 11971958

  • Supervised learning from microarray data COMPSTAT 2002: PROCEEDINGS IN COMPUTATIONAL STATISTICS Hastie, T., Tibshirani, R., Narasimhan, B., Chu, G. 2002: 67-77
  • 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

  • Activation of DNA-dependent protein kinase by single-stranded DNA ends JOURNAL OF BIOLOGICAL CHEMISTRY Hammarsten, O., DeFazio, L. G., Chu, G. 2000; 275 (3): 1541-1550

    Abstract

    DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination. The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PK(CS). To define the DNA structure required for kinase activation, we synthesized a series of DNA molecules and tested their interactions with purified DNA-PK(CS). The addition of unpaired single strands to blunt DNA ends increased binding and activation of the kinase. When single-stranded loops were added to the DNA ends, binding was preserved, but kinase activation was severely reduced. Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase. Significantly, short single-stranded oligonucleotides of 3-10 bases were capable of activating DNA-PK(CS). Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA ends. The structure of DNA-PK(CS) contains an open channel large enough for double-stranded DNA and an adjacent enclosed cavity with the dimensions of single-stranded DNA. The data presented here support a model in which duplex DNA binds to the open channel, and a single-stranded DNA end is inserted into the enclosed cavity to activate the kinase.

    View details for Web of Science ID 000084940000005

    View details for PubMedID 10636842

  • Structure of DNA-dependent protein kinase: implications for its regulation by DNA EMBO JOURNAL Leuther, K. K., Hammarsten, O., Kornberg, R. D., Chu, G. 1999; 18 (5): 1114-1123

    Abstract

    DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described.

    View details for Web of Science ID 000079184600003

    View details for PubMedID 10064579

  • 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

  • The use of electrophoretic mobility shift assays to study DNA repair. Methods in molecular biology (Clifton, N.J.) Hwang, B. J., Smider, V., Chu, G. 1999; 113: 103-120

    View details for PubMedID 10443414

  • Failure of hairpin-ended and nicked DNA to activate DNA-dependent protein kinase: Implications for V(D)J recombination MOLECULAR AND CELLULAR BIOLOGY Smider, V., Rathmell, W. K., Brown, G., Lewis, S., Chu, G. 1998; 18 (11): 6853-6858

    Abstract

    V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.

    View details for Web of Science ID 000076512900065

    View details for PubMedID 9774698

  • p48 activates a UV-damaged-DNA binding factor and is defective in xeroderma pigmentosum group E cells that lack binding activity MOLECULAR AND CELLULAR BIOLOGY Hwang, B. J., Toering, S., FRANCKE, U., Chu, G. 1998; 18 (7): 4391-4399

    Abstract

    A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a "hit-and-run" mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin.

    View details for Web of Science ID 000074380100077

    View details for PubMedID 9632823

  • DNA-dependent protein kinase: DNA binding and activation in the absence of Ku PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hammarsten, O., Chu, G. 1998; 95 (2): 525-530

    Abstract

    In mammalian cells, double-strand break repair and V(D)J recombination require DNA-dependent protein kinase (DNA-PK), a serine/threonine kinase that is activated by DNA. DNA-PK consists of a 460-kDa subunit (p460) that contains a putative kinase domain and a heterodimeric subunit (Ku) that binds to double-stranded DNA ends. Previous reports suggested that the activation of DNA-PK requires the binding of Ku to DNA. To investigate this further, p460 and Ku were purified separately to homogeneity. Surprisingly, p460 was capable of binding to DNA in the absence of Ku. The binding of p460 to double-stranded DNA ends was salt-labile and could be disrupted by single-stranded or supercoiled DNA, properties distinct from the binding of Ku to DNA. Under low salt conditions, which permitted the binding of p460 to DNA ends, the kinase was activated. Under higher salt conditions, which inhibited the binding of p460, activation of the kinase required the addition of Ku. Significantly, when the length of DNA decreased to 22 bp, Ku competed with p460 for DNA binding and inhibited kinase activity. These data demonstrate that p460 is a self-contained kinase that is activated by direct interaction with double-stranded DNA and that the role of Ku is to stabilize the binding of p460 to DNA ends.

    View details for Web of Science ID 000071606000018

    View details for PubMedID 9435225

  • Double strand break repair JOURNAL OF BIOLOGICAL CHEMISTRY Chu, G. 1997; 272 (39): 24097-24100

    View details for Web of Science ID A1997XY51500001

    View details for PubMedID 9305850

  • The end-joining reaction in V(D)J recombination. Seminars in immunology Smider, V., Chu, G. 1997; 9 (3): 189-197

    Abstract

    V(D)J recombination consists of a DNA cleavage reaction catalysed by RAG1 and RAG2, followed by an end-joining reaction that utilizes the cell's double-strand break repair machinery. Genes essential for the end-joining reaction include: XRCC4 encoding a protein of unknown enzymatic function; XRCC5 and XRCC6 encoding 86 and 70 kDa subunits of the Ku autoantigen, a DNA end-binding protein that is also the regulatory subunit of DNA-dependent protein kinase (DNA-PK); and XRCC7 encoding the catalytic subunit (DNA-PKcs) of DNA-PK. Recent progress in understanding the cleavage reaction, coupled with what was previously known about Ku, DNA-PK, and double-strand break repair, provide the foundation for a working model of how V(D)J recombination might be catalysed.

    View details for PubMedID 9200330

  • DNA-dependent protein kinase is not required for accumulation of p53 or cell cycle arrest after DNA damage CANCER RESEARCH Rathmell, W. K., Kaufmann, W. K., Hurt, J. C., Byrd, L. L., Chu, G. 1997; 57 (1): 68-74

    Abstract

    In response to DNA damage, cells transduce a signal that leads to accumulation and activation of p53 protein, transcriptional induction of several genes, including p21, gadd45, and gadd153, and cell cycle arrest. One hypothesis is that the signal is mediated by DNA-dependent protein kinase (DNA-PK), which consists of a catalytic subunit (DNA-PKcs) and a regulatory subunit (Ku). DNA-PK has several characteristics that support this hypothesis: Ku binds to DNA damaged by nicks or double-strand breaks, DNA-PKcs is activated when Ku binds to DNA, DNA-PK will phosphorylate p53 and other cell cycle regulatory proteins in vitro, and DNA-PKcs shares homology with ATM, which is mutated in ataxia telangiectasia and involved in signaling the p53 response to ionizing radiation. The hypothesis was tested by analyzing early passage fibroblasts from severe combined immunodeficient mice, which are deficient in DNA-PK. After exposure to ionizing radiation, UV radiation, or methyl methane-sulfonate, severe combined immunodeficient and wild-type cells were indistinguishable in their response. The accumulation of p53, induction of p21, gadd45, and gadd153, and arrest of the cell cycle in G1 and G2 occurred normally. Therefore, DNA-PK is not required for the p53 response or cell cycle arrest after DNA damage.

    View details for Web of Science ID A1997WA69100016

    View details for PubMedID 8988043

  • Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy: Do the genes explain the diseases? TRENDS IN GENETICS Chu, G., Mayne, L. 1996; 12 (5): 187-192

    Abstract

    Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three distinct human syndromes associated with sensitivity to ultraviolet radiation. We review evidence that these syndromes overlap with each other and arise from mutations in genes involved in nucleotide-excision repair and RNA transcription. Attempts have been made to explain the syndromes in terms of defects in repair and transcription. These two biochemical pathways do not easily account for all the features of the syndromes. Therefore, we propose a third pathway, in which the syndromes are due, in part, to defects in a demethylation mechanism involving the excision of methylated cytosine. Perturbation of demethylation could affect the developmentally regulated expression of some genes.

    View details for Web of Science ID A1996UH99800008

    View details for PubMedID 8984734

  • Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants MOLECULAR AND CELLULAR BIOLOGY Errami, A., Smider, V., Rathmell, W. K., He, D. M., Hendrickson, E. A., Zdzienicka, M. Z., Chu, G. 1996; 16 (4): 1519-1526

    Abstract

    X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.

    View details for Web of Science ID A1996UB56200025

    View details for PubMedID 8657125

  • Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells MUTATION RESEARCH-DNA REPAIR Hwang, B. J., Liao, J. C., Chu, G. 1996; 362 (1): 105-117

    Abstract

    XPE binding factor (XPE-BF) is deficient in a subset of patients from xeroderma pigmentosum complementation group E. Binding activity copurifies with a 125 kDa polypeptide (p125) that binds to DNA damaged by ultraviolet (UV) radiation and many other agents. We isolated cDNA encoding a polypeptide with a predicted amino acid sequence that matched the sequences of eleven tryptic peptides derived from digestion of XPE-BF purified from human placenta. In vitro transcription and translation of the cDNA yielded a polypeptide of 125 kDa that bound specifically to UV-damaged DNA. Therefore the cDNA encodes either all or the major component of XPE-BF.

    View details for Web of Science ID A1996TP86900012

    View details for PubMedID 8538642

  • Role of the Ku autoantigen in V(D)J recombination and double-strand break repair MOLECULAR ANALYSIS OF DNA REARRANGEMENTS IN THE IMMUNE SYSTEM Chu, G. 1996; 217: 113-132

    View details for Web of Science ID A1996BJ66K00008

    View details for PubMedID 8787621

  • A NOVEL ROLE FOR DNA PHOTOLYASE - BINDING TO DNA DAMAGED BY DRUGS IS ASSOCIATED WITH ENHANCED CYTOTOXICITY IN SACCHAROMYCES-CEREVISIAE MOLECULAR AND CELLULAR BIOLOGY Fox, M. E., Feldman, B. J., Chu, G. 1994; 14 (12): 8071-8077

    Abstract

    DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.

    View details for Web of Science ID A1994PV67400036

    View details for PubMedID 7969145

  • RESTORATION OF X-RAY RESISTANCE AND V(D)J RECOMBINATION IN MUTANT-CELLS BY KU CDNA SCIENCE Smider, V., Rathmell, W. K., Lieber, M. R., Chu, G. 1994; 266 (5183): 288-291

    Abstract

    Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen. Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage.

    View details for Web of Science ID A1994PM13400038

    View details for PubMedID 7939667

  • XERODERMA-PIGMENTOSUM GROUP-E BINDING-FACTOR RECOGNIZES A BROAD-SPECTRUM OF DNA-DAMAGE MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS Payne, A., Chu, G. 1994; 310 (1): 89-102

    Abstract

    Xeroderma pigmentosum complementation group E binding factor (XPE-BF) is a damaged DNA binding protein that is deficient in a subset of patients from complementation group E of xeroderma pigmentosum. The protein recognizes various forms of DNA damage including some cyclobutane pyrimidine dimers, 6-4 photoproducts, cis-diamminedichloroplatinum(II) adducts, and single-stranded DNA. We now show that it also recognizes damage induced by nitrogen mustard; N-methyl-N'-nitro-N-nitrosoguanidine, and depurination, but has no detectable affinity for DNA adducts generated by trans-diamminedichloroplatinum(II), 4-nitroquinoline-N-oxide, 8-methoxypsoralen, or enzymatically methylated cytosine and adenine. The failure to recognize 4-nitroquinoline-N-oxide and 8-methoxypsoralen adducts is consistent with previous reports that XPE cells carry out wild-type levels of repair synthesis after DNA damage by those drugs. These results demonstrate that XPE-BF is a versatile damage recognition protein, but suggest that other proteins must contribute to the recognition of DNA lesions for the human excision repair pathway.

    View details for Web of Science ID A1994PM08100009

    View details for PubMedID 7523888

  • INVOLVEMENT OF THE KU AUTOANTIGEN IN THE CELLULAR-RESPONSE TO DNA DOUBLE-STRAND BREAKS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rathmell, W. K., Chu, G. 1994; 91 (16): 7623-7627

    Abstract

    The Ku autoantigen is a well-characterized heterodimer of 70 and 86 kDa that binds to DNA ends, but its cellular function has been obscure. An electrophoretic mobility-shift assay and Ku antisera were used to show that Ku or a closely related protein was deficient in three mutant hamster cell lines from x-ray-sensitive complementation group 5, which is characterized by defects in DNA double-strand break repair and V(D)J recombination. Furthermore, Ku protein expression was restored when the cells reverted to x-ray resistance. The Ku p86 gene maps to human chromosome 2q33-35, and group 5 cells are rescued by almost precisely the same region, 2q34-36. Thus, biochemical and genetic evidence suggests that Ku is involved in pathways for DNA recombination and repair. By its association with a DNA-dependent protein kinase activated by DNA ends, Ku may also initiate a signaling pathway induced by DNA damage, perhaps for cell cycle arrest.

    View details for Web of Science ID A1994PA37600051

    View details for PubMedID 8052631

  • A DNA END-BINDING FACTOR INVOLVED IN DOUBLE-STRAND BREAK REPAIR AND V(D)J RECOMBINATION MOLECULAR AND CELLULAR BIOLOGY Rathmell, W. K., Chu, G. 1994; 14 (7): 4741-4748

    Abstract

    We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends but had no detectable affinity for single-stranded DNA ends. Since X rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray-sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DEB factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination. The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (F. Pergola, M. Z. Zdzienicka, and M. R. Lieber, Mol. Cell. Biol. 13:3464-3471, 1993, and G. Taccioli, G. Rathbun, E. Oltz, T. Stamato, P. Jeggo, and F. Alt, Science 260:207-210, 1993), consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.

    View details for Web of Science ID A1994NT62600044

    View details for PubMedID 7516471

  • CELLULAR-RESPONSES TO CISPLATIN - THE ROLES OF DNA-BINDING PROTEINS AND DNA-REPAIR JOURNAL OF BIOLOGICAL CHEMISTRY Chu, G. 1994; 269 (2): 787-790

    Abstract

    The anticancer drug cisplatin provokes a complex response in the cell. A lethal dose of the drug kills cells primarily by forming DNA adducts, causing G2 arrest in the cell cycle, and then triggering apoptosis. A sublethal dose induces drug resistance by several mechanisms, including changes in drug uptake and efflux, glutathione and metallothionein levels, and DNA repair. Cisplatin-DNA adducts bind several cellular proteins, including some that enhance survival of the cell by mediating DNA repair and others that hasten its death by conferring sensitivity to the drug.

    View details for Web of Science ID A1994MR22000001

    View details for PubMedID 8288625

  • MASSIVE CISPLATIN OVERDOSE BY ACCIDENTAL SUBSTITUTION FOR CARBOPLATIN - TOXICITY AND MANAGEMENT CANCER Chu, G., MANTIN, R., Shen, Y. M., BASKETT, G., Sussman, H. 1993; 72 (12): 3707-3714

    Abstract

    Unlike the related drug carboplatin, cisplatin is highly nephrotoxic and must be given with vigorous intravenous hydration at a much lower dose. As the result of an accidental substitution of cisplatin for carboplatin, a 68-year-old woman received a massive overdose of cisplatin without intravenous hydration.Laboratory documentation included measurements of platinum concentrations by atomic absorption spectroscopy and of xeroderma pigmentosum group E (XPE) binding factor, a protein that is involved in the recognition step of DNA repair.Toxicities included severe emesis, myelosuppression, renal failure, and deafness, which are well known. Other toxicities were seizures, hallucinations, loss of vision, and hepatic toxicity, which were unusual and may have been caused by the magnitude of the overdose. As late as day 19, there was a continued cellular response from cisplatin, as evidenced by decreased levels of XPE binding factor in extracts from the patient's peripheral blood lymphocytes. Plasmapheresis was effective in lowering the platinum concentration from greater than 2900 ng/ml to 200 ng/ml and appeared to be of clinical benefit. Even after the onset of renal failure, hydration to increase urine volume resulted in increased urinary excretion of platinum. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to ameliorate myelosuppression. The patient received a transplanted kidney from her monozygotic twin sister and survived with no clinically significant deficit except for deafness.No previous reports exist of survival after such a high dose of cisplatin without intravenous hydration. In the future, patients may benefit from similar management and heightened awareness of the possibility of accidental substitution.

    View details for Web of Science ID A1993ML34100023

    View details for PubMedID 8252487

  • PURIFICATION AND CHARACTERIZATION OF A HUMAN PROTEIN THAT BINDS TO DAMAGED DNA BIOCHEMISTRY Hwang, B. J., Chu, G. 1993; 32 (6): 1657-1666

    Abstract

    Xeroderma pigmentosum (XP) is an inherited disease characterized by defective repair of DNA damaged by ultraviolet (UV) radiation or agents that produce bulky DNA adducts. Human cells contain a factor that is deficient in a subset of patients from XP complementation group E and binds to DNA damaged by UV, cisplatin, or denaturation. This factor, XPE binding factor (XPE-BF), was purified to near homogeneity. The denatured protein migrated as a 125-kDa polypeptide on SDS-PAGE, and the native protein migrated primarily as a monomer on gel filtration and glycerol gradient sedimentation. Sedimentation revealed major peak in binding activity at 6.8 S, corresponding to the monomeric form, and a minor peak at 14.5 S, suggesting a homodimeric form. Binding activity was dependent on unmodified cysteine residues, stimulated by magnesium, and inhibited by zinc. Binding to UV-damaged nucleotides was 500,000-fold greater than for intact nucleotides, explaining how a molecule with an abundance of only 1-2 molecules per megabase can survey the genome for damaged DNA. Binding required a minimal DNA substrate of between 16 and 26 bp, as determined by a novel "shoe size" assay. Consistent with its previously noted versatility, XPE-BF bound to some cyclobutane pyrimidine dimers and at least one other UV-induced lesion. However, it may not bind to a subset of cyclobutane dimers, likely including the thymine dimer. These findings may explain the relatively mild phenotype of XP group E and suggest the existence of at least one other binding protein involved in the XP repair pathway.

    View details for Web of Science ID A1993KM70300033

    View details for PubMedID 8431446

  • BAG MODEL FOR DNA MIGRATION DURING PULSED-FIELD ELECTROPHORESIS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chu, G. 1991; 88 (24): 11071-11075

    Abstract

    A model for pulsed-field electrophoresis was developed by picturing large DNA as a deformable "bag" that (i) moves with limiting mobility in a continuous electric field, (ii) adopts an orientation aligned with the field direction, and (iii) reorients after a change in field direction in a size-dependent manner. The model correctly predicted the resolution of large DNA in a pulsed field including the surprising phenomena of mobility inversion, lateral band spreading, and improved resolution for obtuse angles. A simple parametrization agreed with observations of two completely different aspects of DNA behavior: bulk mobility as measured during gel electrophoresis and molecular reorientation as measured by linear dichroism. The model also provides quantitative guidelines for setting experimental parameters in pulsed-field electrophoresis experiments.

    View details for Web of Science ID A1991GV87700019

    View details for PubMedID 1763022

  • PULSED-FIELD ELECTROPHORESIS OF MEGABASE-SIZED DNA MOLECULAR AND CELLULAR BIOLOGY Gunderson, K., Chu, G. 1991; 11 (6): 3348-3354

    Abstract

    Success in constructing a physical map of the human genome will depend on two capabilities: rapid resolution of very large DNA and identification of migration anomalies. To address these issues, a systematic exploration of pulsed-field electrophoresis conditions for separating multimegabase-sized DNA was undertaken. Conditions were found for first liberating and then separating DNA up to 6 megabases at higher field strengths and more rapidly than previously reported. In addition, some conditions for transversely pulsed fields produced mobility inversion, in which increased size was accompanied by faster rather than slower migration. Importantly, anomalous migration could be identified by the presence of lateral band spreading, in which the DNA band remained sharply defined but spread laterally while moving down the gel. These results have implications for both practical applications and theoretical models of pulsed-field electrophoresis.

    View details for Web of Science ID A1991FM85200048

    View details for PubMedID 2038337

  • SEPARATION OF LARGE DNA BY A VARIABLE-ANGLE CONTOUR-CLAMPED HOMOGENEOUS ELECTRIC-FIELD APPARATUS ANALYTICAL BIOCHEMISTRY Chu, G., Gunderson, K. 1991; 194 (2): 439-446

    Abstract

    A device for separating large DNA molecules by pulsed field electrophoresis is described. Based on the principles of contour-clamped homogeneous electric fields (CHEF), it uses feedback to clamp voltages in a square electrode array, which is compact and inexpensive to construct, adaptable to computer control, and reorients the electric field by arbitrary angles. To illustrate its capabilities, pulsed fields with reorientation angles ranging from 90 to 140 degrees were used to separate DNAs of 4.7 and 5.7 megabases by up to four band-widths in 20 h. The combination of accessible technology and complete control of the electric field should facilitate the search for ways to resolve even larger DNA.

    View details for Web of Science ID A1991FJ17400032

    View details for PubMedID 1862945

  • CISPLATIN-RESISTANT CELLS EXPRESS INCREASED LEVELS OF A FACTOR THAT RECOGNIZES DAMAGED DNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chu, G., Chang, E. 1990; 87 (9): 3324-3327

    Abstract

    Cancer treatment with the drug cisplatin is often thwarted by the emergence of drug-resistant cells. To study this phenomenon, we identified two independent cellular factors that recognize cisplatin-damaged DNA. One of the two factors, designated XPE binding factor, is deficient in complementation group E of xeroderma pigmentosum, an inherited disease characterized by defective repair of DNA damaged by ultraviolet radiation, cisplatin, and other agents. Human tumor cell lines selected for resistance to cisplatin showed more efficient DNA repair and increased expression of XPE binding factor. These results suggest that XPE binding factor may be responsible, at least in part, for the development of cisplatin resistance in human tumors and that the mechanism may be increased DNA repair.

    View details for Web of Science ID A1990DB95900015

    View details for PubMedID 2333286

  • XERODERMA PIGMENTOSUM GROUP-E CELLS LACK A NUCLEAR FACTOR THAT BINDS TO DAMAGED DNA SCIENCE Chu, G., Chang, E. 1988; 242 (4878): 564-567

    Abstract

    The disease xeroderma pigmentosum is characterized by deficient repair of damaged DNA. Fusions of cells from different patients have defined nine genetic complementation groups (A through I), implying that DNA repair in humans involves multiple gene products. In this report, an extension of the gel electrophoresis binding assay was used to identify at least one nuclear factor that (i) bound to DNA damaged by ultraviolet radiation or the antitumor drug cisplatin, but (ii) was notably absent in cells from complementation group E. Therefore, the factor appears to participate in a versatile DNA repair pathway at the stage of binding and recognition.

    View details for Web of Science ID A1988Q634500034

    View details for PubMedID 3175673

Conference Proceedings


  • Toxicity from radiation therapy associated with abnormal transcriptional responses to DNA damage Rieger, K. E., Hong, W. J., Tusher, V. G., Tang, J., Tibshirani, R., Chu, G. ELSEVIER IRELAND LTD. 2004: S29-S29

Footer Links:

Stanford Medicine Resources: