Bio

Academic Appointments


Research & Scholarship

Current Research and Scholarly Interests


Our major focus is to understand the molecular mechanisms involved in maintaining genome integrity during chromosome replication. Our current research programs are:

(1). We investigate what types of mutation in genes that play a critical role in DNA replication can cause an early event in tumorigenesis and are a source of the genetic instability observed in cancer cells. We use both budding and fission yeast as the model organisms to identify replication mutators. Similar and identical mutations are then introduced into homologues genes in human cell lines. We then investigate the physiological effects of the replication mutators on human cells by cytogenetic, cell biologic, and biochemical approaches to resolve the molecular mechanisms that cause the aberrant phenotype in human cells.

(2). We investigate how cells respond to replication stress to maintain genome integrity by checkpoint mechanisms. We used fission yeast as the model organism and replication mutants to induce stress in S-phase. We then investigate how the checkpoint mechanisms maintain genome integrity by inducing tolerance of the replication stress, preventing replication fork collapse, and promoting replication fork re-start. Knowledge gained from the fission yeast studies is then apply to investigate how mammalian cells respond to replication stress to maintain genome integrity.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • Replication-compromised cells require the mitotic checkpoint to prevent tetraploidization CHROMOSOMA Zhang, Z., Arora, S., Zhou, y., Cherry, A., Wang, T. S. 2011; 120 (1): 73-82

    Abstract

    Replication stress often induces chromosome instability. In this study, we explore which factors in replication-compromised cells promote abnormal chromosome ploidy. We expressed mutant forms of either polymerase ? (Pol?) or polymerase ? (Pol?) in normal human fibroblasts to compromise DNA replication. Cells expressing the mutant Pol?-protein failed to sustain mitotic arrest and, when propagated progressively, down-regulated Mad2 and BubR1 and accumulated 4N-DNA from the 2N-DNA cells. Significantly, a population of these cells became tetraploids. The Pol? mutant expressing cells also exhibited elevated cellular senescence markers, suggesting as a mechanism to limit proliferation of the tetraploids. Expression of the Pol? mutant also caused cells to accumulate 4N-DNA. In contrast to the Pol? mutant expressing cells, the Pol? mutant expressing cells expressed sufficient levels of Mad2, BubR1, and cyclin B1 to sustain mitotic arrest, and these cells had normal chromosome ploidy. Together, these results suggest that replication-compromised cells depend on the mitotic checkpoint to prevent mitotic slippage that could result in tetraploidization.

    View details for DOI 10.1007/s00412-010-0292-7

    View details for Web of Science ID 000286628800006

    View details for PubMedID 20827484

  • Rad3-dependent phosphorylation of the checkpoint clamp regulates repair-pathway choice NATURE CELL BIOLOGY Kai, M., Furuya, K., Paderi, F., Carr, A. M., Wang, T. S. 2007; 9 (6): 691-U148

    Abstract

    When replication forks collapse, Rad3 phosphorylates the checkpoint-clamp protein Rad9 in a manner that depends on Thr 225, a residue within the PCNA-like domain. The physiological function of Thr 225-dependent Rad9 phosphorylation, however, remains elusive. Here, we show that Thr 225-dependent Rad9 phosphorylation by Rad3 regulates DNA repair pathways. A rad9(T225C) mutant induces a translesion synthesis (TLS)-dependent high spontaneous mutation rate and a hyper-recombination phenotype. Consistent with this, Rad9 coprecipitates with the post-replication repair protein Mms2. This interaction is dependent on Rad9 Thr 225 and is enhanced by DNA damage. Genetic analyses indicate that Thr 225-dependent Rad9 phosphorylation prevents inappropriate Rhp51-dependent recombination, potentially by redirecting the repair through a Pli1-mediated sumoylation pathway into the error-free branch of the Rhp6 repair pathway. Our findings reveal a new mechanism by which phosphorylation of Rad9 at Thr 225 regulates the choice of repair pathways for maintaining genomic integrity during the cell cycle.

    View details for DOI 10.1038/ncb1600

    View details for Web of Science ID 000246922700015

    View details for PubMedID 17515930

  • Rad4(TopBP1) associates with Srr2, an Spc1 MAPK-regulated protein, in response to environmental stress JOURNAL OF BIOLOGICAL CHEMISTRY Taricani, L., Wang, T. S. 2007; 282 (12): 8793-8800

    Abstract

    Rad4(TopBP1) is a scaffold in a protein complex containing both replication proteins and checkpoint proteins and plays essential roles in both replication and checkpoint responses. We have previously identified four novel fission yeast mutants of rad4+(TopBP1) to explore how Rad4(TopBP1), a single protein, can play multiple roles in genomic integrity maintenance. Among the four novel mutants, rad4-c17(TopBP1) is a thermosensitive mutant. Here, we characterized rad4-c17(TopBP1) and identified a rad4-c17(TopBP1) allele specific suppressor named srr2+ (suppressor of Rad4(TopBP1) R2 domain). srr2+ has previously been identified as an environmental stress-responsive gene (GenBank accession number AL049644.1, locus spcc191.01). srr2+ null cells are sensitive to hydroxyurea (HU) at elevated temperatures. Deletion of srr2+ in rad4-c17(TopBP1) exacerbates the HU sensitivity of the mutant. Overexpression of srr2+ suppresses the rad4-c17(TopBP1) mutant sensitivity to temperature and HU and restores the compromised ability of rad4-c17(TopBP1) to activating Cds1 kinase in response to HU treatment. Furthermore, stress-activated MAPK, Spc1 (also known as StyI or Phh1), induces the expression and phosphorylation of the Srr2 protein. Significantly, environmental stress induces co-precipitation of Srr2 protein with Rad4(TopBP1), and the co-precipitation is compromised in the rad4-c17(TopBP1) mutant. These results have led us to propose a model; Rad4(TopBP1) exists in a large protein complex to coordinate genomic perturbations with checkpoint responses to maintain genomic integrity. In addition, when cells experience environmental stress, Rad4(TopBP1) associates with Srr2, an Spc1 MAPK-responsive protein, to survive the stress, potentially by providing a link of the Spc1 MAPK response to checkpoint responses.

    View details for DOI 10.1074/jbc.M609282200

    View details for Web of Science ID 000245780300026

    View details for PubMedID 17272281

  • Rad4(TopBP1), a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication MOLECULAR BIOLOGY OF THE CELL Taricani, L., Wang, T. S. 2006; 17 (8): 3456-3468

    Abstract

    Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.

    View details for DOI 10.1091/mbc.E06-01-0056

    View details for Web of Science ID 000239502200011

    View details for PubMedID 16723501

  • Methods for studying mutagenesis and checkpoints in Schizosaccharomyces pombe DNA REPAIR, PT B Kai, M., Taricani, L., Wang, T. S. 2006; 409: 183-194

    Abstract

    Mutations in genome caretaker genes can induce genomic instability, which are potentially early events in tumorigenesis. Cells have evolved biological processes to cope with the genomic insults. One is a multifaceted response, termed checkpoint, which is a network of signaling pathways to coordinate cell cycle transition with DNA repair, activation of transcriptional programs, and induction of tolerance of the genomic perturbations. When genomic perturbations are beyond repair, checkpoint responses can also induce apoptosis or senescence to eliminate those deleterious damaged cells. Fission yeast, Schizosaccharomyces pombe (S. pombe) has served as a valuable model organism for studies of the checkpoint signaling pathways. In this chapter, we describe methods used to analyze mutagenesis and recombinational repair induced by genomic perturbations, and methods used to detect the checkpoint responses to replication stress and DNA damage in fission yeast cells. In the first section, we present methods used to analyze the mutation rate, mutation spectra, and recombinational repair in fission yeast when replication is perturbed by either genotoxic agents or mutations in genomic caretaker gene such as DNA replication genes. In the second section, we describe methods used to examine checkpoint activation in response to chromosome replication stress and DNA damage. In the final section, we comment on how checkpoint activation regulates mutagenic synthesis by a translesion DNA polymerase in generating a mutator phenotype of small sequence alterations in cells, and how a checkpoint kinase appropriately regulates an endonuclease complex to either prevent or allow deletion of genomic sequences and recombinational repair when fission yeast cells experience genomic perturbation in order to avoid deleterious mutations and maintain cell growth.

    View details for DOI 10.1016/S0076-6879(05)09011-7

    View details for Web of Science ID 000238354800011

    View details for PubMedID 16793402

  • Replication checkpoint kinase Cds1 regulates Mus81 to reserve genome integrity during replication stress GENES & DEVELOPMENT Kai, M., Boddy, M. N., Russell, P., Wang, T. S. 2005; 19 (8): 919-932

    Abstract

    The replication checkpoint kinase Cds1 preserves genome integrity by stabilizing stalled replication forks. Cds1 targets substrates through its FHA domain. The Cds1 FHA domain interacts with Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease. We report here that Mus81 and Rhp51 are required for generating deletion mutations in fission yeast replication mutants that experience replication stress. A mutation in the Mus81 FHA-binding motif eliminates its Cds1-binding and Cds1-dependent phosphorylation. Furthermore, this mutation exacerbates the deletion mutator phenotype of a replication mutant, and induces a hyper-recombination phenotype in hydroxyurea-treated cells. In unperturbed cells, Mus81 associates with chromatin throughout S phase. In replication mutants grown at semipermissive temperature, Mus81 undergoes minor Cds1-dependent phosphorylation, remains chromatin-associated, generates deletion mutations, and maintains cell growth. Upon S-phase arrest by acute hydroxyurea treatment, Mus81 is not required for cell viability but is essential for recovery from replication fork collapse. Moreover, Mus81 undergoes extensive Cds1-dependent phosphorylation and dissociates from chromatin in hydroxyurea-arrested cells, thereby preventing it from cleaving stalled replication forks that could lead to fork breakage and chromosomal rearrangement. These results provide novel insights into how Cds1 regulates Mus81 accordingly when cells experience different replication stress to preserve genome integrity.

    View details for DOI 10.1101/gad.1304305

    View details for Web of Science ID 000228463300005

    View details for PubMedID 15805465

  • A coordinated temporal interplay of nucleosome reorganization factor, sister chromatin cohesion factor, and DNA polymerase alpha facilitates DNA replication MOLECULAR AND CELLULAR BIOLOGY Zhou, Y. J., Wang, T. S. 2004; 24 (21): 9568-9579

    Abstract

    DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase alpha (Polalpha). The catalytic subunit of budding yeast Polalpha (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polalpha (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polalpha protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

    View details for DOI 10.1128/MCB.24.21.9568-9579.2004

    View details for Web of Science ID 000224943300028

    View details for PubMedID 15485923

  • The B-subunit of DNA polymerase alpha-primase associates with the origin recognition complex for initiation of DNA replication MOLECULAR AND CELLULAR BIOLOGY Uchiyama, M., Wang, T. S. 2004; 24 (17): 7419-7434

    Abstract

    The B-subunit (p70/Pol12p) of the DNA polymerase alpha-primase (Polalpha-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polalpha-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polalpha-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polalpha-primase in the initiation of both leading and lagging strands at the replication origins.

    View details for DOI 10.1128/MCB.24.17.7419-7434.2004

    View details for Web of Science ID 000223443800010

    View details for PubMedID 15314153

  • Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS Kai, M., Wang, T. S. 2003; 532 (1-2): 59-73

    Abstract

    Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

    View details for DOI 10.1016/j.mrfmmm.2003.08.010

    View details for Web of Science ID 000187355500006

    View details for PubMedID 14643429

  • Genomic instability induced by mutations in Saccharomyces cerevisiae POL1 GENETICS Gutierrez, P. J., Wang, T. S. 2003; 165 (1): 65-81

    Abstract

    Mutations of chromosome replication genes can be one of the early events that promote genomic instability. Among genes that are involved in chromosomal replication, DNA polymerase alpha is essential for initiation of replication and lagging-strand synthesis. Here we examined the effect of two mutations in S. cerevisiae POL1, pol1-1 and pol1-17, on a microsatellite (GT)(16) tract. The pol1-17 mutation elevated the mutation rate 13-fold by altering sequences both inside and downstream of the (GT)(16) tract, whereas the pol1-1 mutation increased the mutation rate 54-fold by predominantly altering sequences downstream of the (GT)(16) tract in a RAD52-dependent manner. In a rad52 null mutant background pol1-1 and pol1-17 also exhibited different plasmid and chromosome loss phenotypes. Deletions of mismatch repair (MMR) genes induce a differential synergistic increase in the mutation rates of pol1-1 and pol1-17. These findings suggest that perturbations of DNA replication in these two pol1 mutants are caused by different mechanisms, resulting in various types of mutations. Thus, mutations of POL1 can induce a variety of mutator phenotypes and can be a source of genomic instability in cells.

    View details for Web of Science ID 000185618200006

    View details for PubMedID 14504218

  • Replication proteins influence the maintenance of telomere length and telomerase protein stability MOLECULAR AND CELLULAR BIOLOGY Dahlen, M., Sunnerhagen, P., Wang, T. S. 2003; 23 (9): 3031-3042

    Abstract

    We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells. Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.

    View details for DOI 10.1128/MCB.23.9.3031-3042.2003

    View details for Web of Science ID 000182325500001

    View details for PubMedID 12697806

  • Checkpoint activation regulates mutagenic translesion synthesis GENES & DEVELOPMENT Kai, M., Wang, T. S. 2003; 17 (1): 64-76

    Abstract

    Cells have evolved checkpoint responses to arrest or delay the cell cycle, activate DNA repair networks, or induce apoptosis after genomic perturbation. Cells have also evolved the translesion synthesis processes to tolerate genomic lesions by either error-free or error-prone repair. Here, we show that after a replication perturbation, cells exhibit a mutator phenotype, which can be significantly affected by mutations in the checkpoint elements Cds1 and Rad17 or translesion synthesis polymerases DinB and Polzeta. Cells respond to genomic perturbation by up-regulation of DinB in a checkpoint activation-dependent manner. Moreover, association of DinB with chromatin is dependent on functional Rad17, and DinB physically interacts with the checkpoint-clamp components Hus1 and Rad1. Thus, translesion synthesis is a part of the checkpoint response.

    View details for DOI 10.1101/gad.1043203

    View details for Web of Science ID 000180280700007

    View details for PubMedID 12514100

  • Fission yeast Rad17 associates with chromatin in response to aberrant genomic structures MOLECULAR AND CELLULAR BIOLOGY Kai, M., Tanaka, H., Wang, T. S. 2001; 21 (10): 3289-3301

    Abstract

    Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1Delta or rad3Delta cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1Delta are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys(118) of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in the rad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.

    View details for Web of Science ID 000168306700002

    View details for PubMedID 11313455

  • Role of fission yeast primase catalytic subunit in the replication checkpoint MOLECULAR BIOLOGY OF THE CELL Griffiths, D. J., Liu, V. F., Nurse, P., Wang, T. S. 2001; 12 (1): 115-128

    Abstract

    To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

    View details for Web of Science ID 000166353800010

    View details for PubMedID 11160827

  • Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation MOLECULAR AND CELLULAR BIOLOGY Tan, S. Y., Wang, T. S. 2000; 20 (21): 7853-7866

    Abstract

    To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations of Schizosaccharomyces pombe DNA primase. DNA primase has two subunits, Spp1 and Spp2 (S. pombe primases 1 and 2). Spp1 is the catalytic subunit that synthesizes the RNA primer, which is then extended by DNA polymerase alpha (Polalpha) to synthesize an initiation DNA structure, and this catalytic function of Polalpha is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Polalpha. Thermosensitive mutations of spp2(+) destabilize the Polalpha-primase complex, resulting in an allele-specific S phase checkpoint defect. The mutant exhibiting a more severe checkpoint defect also has a higher extent of Polalpha-primase complex instability and deficiency in the hydroxyurea-induced Cds1-mediated intra-S phase checkpoint response. However, this mutant is able to activate the Cds1 response to S phase arrest induced by temperature. These findings suggest that the Cds1 response to the S-phase arrest signal(s) induced by a initiation mutant is different from that induced by hydroxyurea. Interestingly, a polalphats mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Polalpha-primase complex stability, and the Cds1 response induced by hydroxyurea or by the mutant arrests at the restrictive temperature. Thus, the Cds1-mediated intra-S phase checkpoint response induced by hydroxyurea can also be distinguished from the S-M phase checkpoint response that requires the initiation DNA synthesis by Polalpha.

    View details for Web of Science ID 000089794200003

    View details for PubMedID 11027257

  • A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints JOURNAL OF CELL SCIENCE Griffiths, D., Uchiyama, M., Nurse, P., Wang, T. S. 2000; 113 (6): 1075-1088

    Abstract

    To further dissect the genetic differences between the checkpoint pathway following S-phase cdc arrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase delta mutant, cdc20-M10. One such checkpoint mutant, rad17-d14, was found to display the cut phenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1 mutant. Unlike chk1 , rad17-d14 was not sensitive to UV irradiation. Interestingly, the ionising radiation sensitivity of rad17-d14 was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G(2) phase. Biochemical analysis attributes the checkpoint defects of rad17-d14 to the failure to phosphorylate the checkpoint effector Chk1p. To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed. Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase. This interaction occurred irrespective of the arrest with cdc20-M10 and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.

    View details for Web of Science ID 000086312400018

    View details for PubMedID 10683155

  • Schizosaccharomyces pombe replication and repair proteins: Proliferating cell nuclear antigen (PCNA) METHODS Arroyo, M. P., Wang, T. S. 1999; 18 (3): 335-348

    Abstract

    Schizosaccharomyces pombe has a cell cycle progression with distinctive phases that serves as a perfect model system for investigating DNA replication and repair of eukaryotic cells. Here, we use proliferating cell nuclear antigen (PCNA) of S. pombe to demonstrate how the function of this protein in both DNA replication and repair can be assessed by genetic and biochemical approaches. We describe a method of introducing site-specific mutations into the fission yeast PCNA gene pcn1(+). The in vivo effects of these pcn1 mutants in a strain with a null pcn1 background are described and their in vitro biochemical properties are characterized. Mutants described here are those that are defective in enhancing processivity of DNA polymerase delta, show temperature-sensitive growth, and have increased sensitivity to hydroxyurea (HU), UV and gamma irradiation, and methyl methanesulfonate (MMS). Three mutants that show reduced growth rate in vivo and decreased capacity to enhance polymerase delta DNA synthetic activity and processivity in vitro-pcn1-1, pcn1-5, and pcn1-26-are described as examples of using a genetic approach to identify the biochemical function of replication proteins. One cold-sensitive growth allele, pcn1-3, that has a recessive cold-sensitive cdc phenotype and shows sensitivity to HU and UV and gamma irradiation is used as an example of using the genetic approach to reveal the function of replication proteins in repair. The power of combining both biochemical and genetic disciplines is emphasized. Methods for site-directed mutagenesis, in vitro analysis of mutant proteins, and in vivo characterization of mutants in response to UV or gamma irradiation, MMS, HU, and temperature, as well as genetic epistasis are described. Locations of functionally significant residues on the PCNA tertiary structure are summarized.

    View details for Web of Science ID 000081752300014

    View details for PubMedID 10454995

  • Mutator phenotype induced by aberrant replication MOLECULAR AND CELLULAR BIOLOGY Liu, V. F., Bhaumik, D., Wang, T. S. 1999; 19 (2): 1126-1135

    Abstract

    We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase delta (poldelta) and mutants of Polalpha, two Poldelta subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2(+), which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Delta background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2Delta was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Delta. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.

    View details for Web of Science ID 000078140900016

    View details for PubMedID 9891047

  • Human papillomavirus DNA replication - Interactions between the viral E1 protein and two subunits of human DNA polymerase alpha/primase JOURNAL OF BIOLOGICAL CHEMISTRY Conger, K. L., Liu, J. S., KUO, S. R., CHOW, L. T., Wang, T. S. 1999; 274 (5): 2696-2705

    Abstract

    Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase alpha/primase (pol alpha/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol alpha/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2-dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.

    View details for Web of Science ID 000078319500018

    View details for PubMedID 9915800

  • Mutational effect of fission yeast Pol alpha on cell cycle events MOLECULAR BIOLOGY OF THE CELL Bhaumik, D., Wang, T. S. 1998; 9 (8): 2107-2123

    Abstract

    Polalpha is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polalpha+. Germinating spores carrying either a deletion of polalpha+ (polalphaDelta) or a structurally intact but catalytically dead polalpha mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polalpha, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polalphats allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polalphats in cdc20 (polepsilon mutant) background arrested with a cdc phenoytpe, not a polalphats-like phenotype. At 25 degrees C, replication perturbation caused by polalphats alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36 degrees C, replication disruption caused by polalphats alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polalpha is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polalphats mutations induce differential downstream kinase responses.

    View details for Web of Science ID 000075348400013

    View details for PubMedID 9693370

  • Mutant PCNA alleles are associated with cdc phenotypes and sensitivity to DNA damage in fission yeast MOLECULAR AND GENERAL GENETICS Arroyo, M. P., Wang, T. S. 1998; 257 (5): 505-518

    Abstract

    Twenty-eight site-directed mutations were introduced into the fission yeast gene (pcn1+) that encodes proliferating cell nuclear antigen (PCNA) and their in vivo effects analyzed in a strain with a null pcn1 background. Mutants defective in enhancing processivity of DNA polymerase delta have previously been identified. In this study, we assessed all of the mutants for their sensitivities to temperature, hydroxyurea, UV irradiation and methyl methanesulfonate (MMS), and specific mutants were also tested for sensitivity to gamma irradiation. One cold-sensitive allele, pcn1-3, was characterized in detail. This mutant had a recessive cold-sensitive cdc phenotype and showed sensitivity to hydroxyurea, UV, and gamma irradiation. At the non-permissive temperature pcn1-3 protein was able to form homotrimers in solution and showed increased stimulation of both synthetic activity and processivity of DNA polymerase delta relative to the wild-type Pcn1+ protein. Epistasis analyses indicated that pcn1-3 is defective in the repair pathway involving rad2+ but not defective in the classical nucleotide excision repair pathway involving rad13+. Furthermore, pcn1-3 is either synthetically or conditionally lethal in null checkpoint rad backgrounds and displays a mitotic catastrophe phenotype in these backgrounds. A model for how pcn1-3 defects may affect DNA repair and replication is presented.

    View details for Web of Science ID 000073027100002

    View details for PubMedID 9563836

  • DNA replication and order of cell cycle events: A role for protein isoprenylation? BIOLOGICAL CHEMISTRY Galli, I., Uchiyama, M., Wang, T. S. 1997; 378 (9): 963-973

    Abstract

    When the aya1+ gene is mutated, Schizosaccharomyces pombe cells become unable to react appropriately to a delay in DNA replication. Instead of stalling the cell cycle to allow completion of DNA synthesis, they proceed unperturbed towards mitosis and attempt to segregate the still unreplicated chromosomes. As a result, the genetic material segregates unevenly and the nuclei assume a mitotic catastrophe phenotype, characterized by torn chromosomes (cut), anucleated cells and scattered chromosomes. Interestingly, the aya1 phenotype can be suppressed by overexpression of either the catalytic subunit of S. pombe DNA polymerase alpha or of a novel protein called hur1 +p. The latter bears significant homology to the core of the human Rab escort protein, which belongs to a family of factors necessary to the post-translational isoprenylation of proteins like Ras, Rab and lamin B. When isoprenylation is chemically inhibited with R-limonene (a monoterpene derived from orange rind), wild type S. pombe cells become insensitive to an S phase delay, in a manner strongly reminiscent of aya1 mutants. Moreover, overexpression of hur1 +p in wild type cells rescues the failing checkpoint function. We propose that there is a strong correlation between the aya1 phenotype, S-M phase checkpoint function, and isoprenylation events in fission yeast.

    View details for Web of Science ID A1997XZ65100004

    View details for PubMedID 9348105

  • A novel mutant allele of Schizosaccharomyces pombe rad26 defective in monitoring S-phase progression to prevent premature mitosis MOLECULAR AND CELLULAR BIOLOGY Uchiyama, M., Galli, I., Griffiths, D. J., Wang, T. S. 1997; 17 (6): 3103-3115

    Abstract

    A semipermissive growth condition was defined for a Schizosaccharomyces pombe strain carrying a thermosensitive allele of DNA polymerase delta (pol delta ts03). Under this condition, DNA polymerase delta is semidisabled and causes a delay in S-phase progression. Using a genetic strategy, we have isolated a panel of mutants that enter premature mitosis when DNA replication is incomplete but which are not defective for arrest in G2/M following DNA damage. We characterized the aya14 mutant, which enters premature mitosis when S phase is arrested by genetic or chemical means. However, this mutant is sensitive to neither UV nor gamma irradiation. Two genomic clones, rad26+ and cds1+, were found to suppress the hydroxyurea sensitivity of the aya14 mutant. Genetic analysis indicates that aya14 is a novel allele of the cell cycle checkpoint gene rad26+, which we have named rad26.a14. cds1+ is a suppressor which suppresses the S-phase feedback control defect of rad26.a14 when S phase is inhibited by either hydroxyurea or cdc22, but it does not suppress the defect when S phase is arrested by a mutant DNA polymerase. Analyses of rad26.a14 in a variety of cdc mutant backgrounds indicate that strains containing rad26.a14 bypass S-phase arrest but not G1 or late S/G2 arrest. A model of how Rad26 monitors S-phase progression to maintain the dependency of cell cycle events and coordinates with other rad/hus checkpoint gene products in responding to radiation damage is proposed.

    View details for Web of Science ID A1997WZ63700015

    View details for PubMedID 9154809

  • Schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA polymerase delta processivity JOURNAL OF BIOLOGICAL CHEMISTRY Arroyo, M. P., Downey, K. M., So, A. G., Wang, T. S. 1996; 271 (27): 15971-15980

    Abstract

    We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu2, Asp63, Arg64, Gly69, Gln201, Glu259, and Glu260, either as single or as double mutants. Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Deltapcna). Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp63, Gln201, Glu259, or Glu260 to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA polymerase delta holoenzyme similar to calf thymus PCNA. Mutations of Leu2 to Val or Arg64 to Ala, either singly or as a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA polymerase delta but showed no deficiency in stimulation of the ATPase activity of replication factor C. S. pombe Deltapcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes. These cells, however, were not sensitive to UV irradiation. Together, these in vitro and in vivo studies suggest that the side chains of Leu2 and Arg64 in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA polymerase delta for processive DNA synthesis during DNA replication.

    View details for Web of Science ID A1996UW35200022

    View details for PubMedID 8663159

  • STUDIES OF SCHIZOSACCHAROMYCES-POMBE DNA-POLYMERASE-ALPHA AT DIFFERENT STAGES OF THE CELL-CYCLE NUCLEIC ACIDS RESEARCH Park, H., Davis, R., Wang, T. S. 1995; 23 (21): 4337-4344

    Abstract

    The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.

    View details for Web of Science ID A1995TG42400017

    View details for PubMedID 7501454

  • MUTATIONAL STUDIES OF HUMAN DNA-POLYMERASE-ALPHA - LYSINE-950 IN THE 3RD MOST CONSERVED REGION OF ALPHA-LIKE DNA-POLYMERASES IS INVOLVED IN BINDING THE DEOXYNUCLEOSIDE TRIPHOSPHATE JOURNAL OF BIOLOGICAL CHEMISTRY Dong, Q., Wang, T. S. 1995; 270 (37): 21563-21570

    Abstract

    The function of a lysine residue, Lys950, of human DNA polymerase alpha located in the third most conserved region and conserved in all of the alpha-like polymerases was analyzed by site-directed mutagenesis. Lys950 was mutagenized to Arg, Ala, or Asn. The mutant enzymes were expressed in insect cells infected with recombinant baculoviruses and purified to near homogeneity. The mutant enzymes had specific activities ranging from 8 to 22% of the wild type. All three Lys950 mutants utilized Mn2+ as metal activator more effectively than the wild type enzyme and showed an increase in Km values for deoxynucleoside triphosphate but not k(cat) values in reactions with either Mg2+ or Mn2+ as the metal activator. Although mutation of the Lys950 residue caused an increase in Km values for deoxynucleoside triphosphates, mutations of Lys950 to Arg, Ala, or Asn did not alter the mutant enzymes' misinsertion efficiency in reactions with Mg2+ as a metal activator as compared with that of the wild type, suggesting that the base of the incoming deoxynucleoside triphosphate is not the structural feature interacting with the Lys950 side chain. In reaction with Mn2+ as a metal activator, all three Lys950 mutants had an improved fidelity for deoxynucleotide misinsertion compared to wild type. Inhibition studies of the three Lys950 mutant derivatives with an inhibitor, structural analogs of deoxynucleoside triphosphate, and pyrophosphate suggest that the deoxyribose sugar and beta-,gamma-phosphate groups are not the structural feature recognized by the Lys950 side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1995RU75700024

    View details for PubMedID 7665569

  • INTERACTION OF HERPES-SIMPLEX VIRUS-1 ORIGIN-BINDING PROTEIN WITH DNA-POLYMERASE-ALPHA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, S. S., Dong, Q., Wang, T. S., Lehman, I. R. 1995; 92 (17): 7882-7886

    Abstract

    The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.

    View details for Web of Science ID A1995RP74800058

    View details for PubMedID 7644508

  • Purification of mammalian DNA polymerases: DNA polymerase alpha DNA REPLICATION Wang, T. S., Copeland, W. C., Rogge, L., Dong, Q. 1995; 262: 77-84

    View details for Web of Science ID A1995BE40P00008

    View details for PubMedID 8594385

  • Rationale for mutagenesis of DNA polymerase active sites: DNA polymerase alpha DNA REPLICATION Copeland, W. C., Dong, Q., Wang, T. S. 1995; 262: 294-303

    View details for Web of Science ID A1995BE40P00023

    View details for PubMedID 8594355

  • DOWN-REGULATION OF GENES ENCODING DNA-REPLICATION PROTEINS DURING CELL-CYCLE EXIT CELL GROWTH & DIFFERENTIATION Moore, A. L., Wang, T. S. 1994; 5 (5): 485-494

    Abstract

    We have studied the expression of genes encoding DNA replication proteins during different cell growth events. Gene expression of human DNA polymerase alpha-DNA primase, a principal chromosomal replication enzyme complex, is up-regulated during the entrance of a cell from quiescence into the mitotic cell cycle. In contrast, expression of these genes is greatly reduced in fibroblasts rendered temporarily quiescent by contact inhibition or serum starvation. In actively cycling cells, DNA polymerase alpha-DNA primase genes are expressed at all stages of the cell cycle. To investigate how their gene expression is regulated in cells permanently exiting the cell cycle during terminal differentiation, we used a novel method to obtain a pure population of such cells. In this report, we describe the down-regulation of gene expression of DNA polymerase alpha during both HL-60 (human myeloid) and MEL (mouse erythroleukemia) cell differentiation. Gene expression of the two subunits of DNA primase, p49 and p58, is also down-regulated at the mRNA level in differentiated MEL cells. In differentiated HL-60 cells, the decline of DNA polymerase alpha gene expression occurs at both the transcript and protein levels. Down-regulation of DNA polymerase alpha at the steady state transcript level is caused, at least in part, by a decreased rate of transcription initiation without transcription elongation block.

    View details for Web of Science ID A1994NK21100003

    View details for PubMedID 8049155

  • ENZYMATIC CHARACTERIZATION OF THE INDIVIDUAL MAMMALIAN PRIMASE SUBUNITS REVEALS A BIPHASIC MECHANISM FOR INITIATION OF DNA-REPLICATION JOURNAL OF BIOLOGICAL CHEMISTRY Copeland, W. C., Wang, T. S. 1993; 268 (35): 26179-26189

    Abstract

    The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58. Neither of the singly purified primase subunits displayed primase activity alone, but the p49 subunit was able to extend a riboprimer, indicating that this subunit contains an RNA polymerase activity. The p58 subunit cooperated with the p49 subunit in binding the initiating purine to form the initial dinucleotide. After initiation, the p49 subunit alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit. The Km(ATP) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the Km(ATP) of the single p49 subunit in a ribo(A) primer extension assay. In addition, labeled ATP cross-linked to both of the individually purified subunits but with a striking difference in affinities; cross-linking was 11-fold more efficient to the p49 subunit. The interaction of the two primase subunits with polymerase alpha was also investigated. Immunoprecipitation experiments indicate that only the p58 subunit directly contacts the p180 subunit of DNA polymerase alpha. Competition experiments in the coupled primase-polymerase assay with a catalytically inactive mutant of DNA polymerase alpha and the Klenow fragment suggest that the DNA polymerase alpha-primase complex does not dissociate from the primer during the transition from RNA to DNA synthesis.

    View details for Web of Science ID A1993MK42500028

    View details for PubMedID 8253737

  • MUTATIONAL STUDIES OF HUMAN DNA POLYMERASE-ALPHA - SERINE 867 IN THE 2ND MOST CONSERVED REGION AMONG ALPHA-LIKE DNA-POLYMERASES IS INVOLVED IN PRIMER BINDING AND MISPAIR PRIMER EXTENSION JOURNAL OF BIOLOGICAL CHEMISTRY Dong, Q., Copeland, W. C., Wang, T. S. 1993; 268 (32): 24175-24182

    Abstract

    The second most conserved region of alpha-like DNA polymerases, region II, spans a block of 40 amino acid residues centered at the core sequence -DFNSLYPSII-. In the previous paper, we described mutational studies of 3 amino acid residues in region II which includes 2 amino acid residues in the core sequence. We showed that residues Asp860 and Tyr865 in the core sequence are involved in substrate deoxynucleotide triphosphate (dNTP) binding. We further showed that the phenyl moiety of the Tyr865 side chain interacts with the incoming dNTP and is responsible for the misinsertion fidelity of the enzyme. In this report, we investigated the function of 2 serine residues, Ser863 and Ser867, in this core sequence. Mutation of these 2 Ser residues to either Ala or Thr yielded mutant enzymes with similar Km for dNTPs, kcat, processivity, and misinsertion fidelity of DNA synthesis as the wild type enzyme. However, mutation of Ser867 to Ala demonstrated a 30-fold increase in Km for primer-template and a 5-fold higher KD for binding primer-template. DNA footprinting experiments of primer with the dideoxynucleotide terminus indicated that the structural feature of the primer recognized by Ser867 is the 3'-OH terminus. Single-stranded DNA inhibition data suggest that removal of the hydroxyl side chain of Ser867 affects the polymerase's interaction with primer and not with template. Mutation of Ser867 to Ala also decreases the mutant enzyme's Km for dNTP to extend a mispaired primer and thus enhances its capacity to extend a mispaired primer terminus. These data support the conclusion that the hydroxyl side chain of Ser867 of human DNA polymerase alpha is involved in primer interaction during DNA synthesis and plays an essential role in mispair extension fidelity of DNA synthesis.

    View details for Web of Science ID A1993MF29400069

    View details for PubMedID 8226964

  • MUTATIONAL STUDIES OF HUMAN DNA POLYMERASE-ALPHA - IDENTIFICATION OF RESIDUES CRITICAL FOR DEOXYNUCLEOTIDE BINDING AND MISINSERTION FIDELITY OF DNA-SYNTHESIS JOURNAL OF BIOLOGICAL CHEMISTRY Dong, Q., Copeland, W. C., Wang, T. S. 1993; 268 (32): 24163-24174

    Abstract

    Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA polymerase alpha catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had polymerase activity ranging from 4 to 60% compared with the wild type polymerase alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their Km values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the Km for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg(2+)-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA polymerase alpha are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in kcat, an increase in Km for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the alpha-like DNA polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the polymerase alpha catalytic site.

    View details for Web of Science ID A1993MF29400068

    View details for PubMedID 8226963

  • IN-VIVO SPECIES-SPECIFICITY OF DNA POLYMERASE-ALPHA MOLECULAR & GENERAL GENETICS Francesconi, S., Copeland, W. C., Wang, T. S. 1993; 241 (3-4): 457-466

    Abstract

    The DNA polymerase alpha enzymes from human, and budding (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are homologous proteins involved in initiation and replication of chromosomal DNA. Sequence comparison of human DNA polymerase alpha with that of S. cerevisiae and S. pombe shows overall levels of amino acid sequence identity of 32% and 34%, respectively. We report here that, despite the sequence conservation among these three enzymes, functionally active human DNA polymerase alpha fails to rescue several different conditional lethal alleles of the budding yeast POL1 gene at nonpermissive temperature. Furthermore, human DNA polymerase alpha cannot complement a null allele of budding yeast POL1 either in germinating spores or in vegetatively growing cells. In fission yeast, functionally active human DNA polymerase alpha is also unable to complement the disrupted pol alpha::ura4+ allele in germinating spores. Thus, in vivo, DNA polymerase alpha has stringent species specificity for initiation and replication of chromosomal DNA.

    View details for Web of Science ID A1993ME53200026

    View details for PubMedID 8246900

  • FISSION YEAST WITH DNA-POLYMERASE DELTA TEMPERATURE-SENSITIVE ALLELES EXHIBITS CELL-DIVISION CYCLE PHENOTYPE NUCLEIC ACIDS RESEARCH Francesconi, S., Park, H., Wang, T. S. 1993; 21 (16): 3821-3828

    Abstract

    DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.

    View details for Web of Science ID A1993LT96500032

    View details for PubMedID 8367300

  • FIDELITY STUDIES OF THE HUMAN DNA POLYMERASE-ALPHA - THE MOST CONSERVED REGION AMONG ALPHA-LIKE DNA-POLYMERASES IS RESPONSIBLE FOR METAL-INDUCED INFIDELITY IN DNA-SYNTHESIS JOURNAL OF BIOLOGICAL CHEMISTRY Copeland, W. C., LAM, N. K., Wang, T. S. 1993; 268 (15): 11041-11049

    Abstract

    Mutational studies in the highly conserved region I domain of the human DNA polymerase alpha enzyme demonstrated a change in metal cation-specific catalysis. Here, we extend the investigation to include the fidelity of DNA synthesis by these mutants, studying misinsertion, mispair extension, and the nucleotide analog utilization. The fidelity of region I mutants and wild type human DNA polymerase alpha enzyme were analyzed with either Mg2+ or Mn2+ as the metal activator. Despite the known mutagenic effect of Mn2+ in causing polymerases to misinsert nucleotides and to utilize dideoxynucleotides, we have found that two region I mutants, D1002N and T1003S, which utilize Mn2+ in catalysis more effectively than Mg2+, actually have a 70- and 40-fold higher misinsertion fidelity, respectively, in Mn(2+)-catalyzed reactions than that of the wild type enzyme. The enhanced misinsertion fidelity of these two mutants in Mn(2+)-catalyzed reactions is due to Km discrimination of the incorrect nucleotide where the D1002N and T1003S had a 850- and 62-fold higher Km for insertion of incorrect than correct nucleotide, respectively. In Mg(2+)-catalyzed reactions, all of the region I mutants exhibited similar misinsertion efficiencies as the wild type polymerase. Study of mispair extension showed that in Mn(2+)-catalyzed ractions, the wild type polymerase alpha enzyme readily extended mispair termini. In contrast, the two region I mutants, D1002N and T1003S, were unable to extend the mispaired termini in either Mg(2+)- or Mn(2+)-catalyzed reactions. These results suggest that the side chains of region I amino acids play an essential role in the Mn(2+)-induced infidelity during DNA synthesis by human DNA polymerase alpha. The effects of the metal activator on the utilization of two nucleotide analogs, 3'-azido-3'-deoxythymidine triphosphate and ddCTP, by the region I mutants were also investigated.

    View details for Web of Science ID A1993LD46600050

    View details for PubMedID 8496165

  • MUTATIONAL ANALYSIS OF THE HUMAN DNA POLYMERASE-ALPHA - THE MOST CONSERVED REGION IN ALPHA-LIKE DNA-POLYMERASES IS INVOLVED IN METAL-SPECIFIC CATALYSIS JOURNAL OF BIOLOGICAL CHEMISTRY Copeland, W. C., Wang, T. S. 1993; 268 (15): 11028-11040

    Abstract

    Five site-directed mutations were introduced at the most conserved amino acids in region I (YGDTDS) of the human DNA polymerase alpha catalytic subunit. Mutant proteins were expressed in the baculovirus system by an improved method and purified by a rapid one-step purification in high yield and high specific activity. The Asp1004 to Asn mutation produced a protein with no detectable polymerase activity while other mutations gave activities from 1 to 20% of the wild type polymerase activity. Steady state kinetic analysis of the active mutants indicates that none of the mutations caused a change in Km(dNTP) or KD(DNA), but all active mutants showed a decrease in kcat. Thus, the effect of these conserved mutations is manifest in altered rates of catalysis. Two mutations, Asp1002 to Asn and Thr1003 to Ser, caused the enzyme to utilize Mn2+ more effectively in catalysis than Mg2+, suggesting that these amino acids are involved in metal binding. Rates of catalysis by the D1002N and T1003S mutants, as well as Y1000F mutant were improved 80-, 30-, and 70-fold, respectively, on homopolymer templates when Mn2+ replaced Mg2+ as the activator metal. The results from these mutational studies suggest that this highly conserved region binds the metal which is essential for catalysis. The Asp1002 may participate directly in chelating the metal. Results from the T1003S mutant suggest that the beta-methyl group of the threonine side chain might be locked in a hydrophobic pocket preventing free rotation around the C alpha-C beta bond, thus positioning the Thr1003 hydroxyl group to form a crucial bond with the metal ion. In addition, D1002N and T1003S displayed a 20-fold resistance to aphidicolin compared to the wild type polymerase alpha, and all of the active mutants displayed altered sensitivity to butylphenyl-dGTP. Models of the involvement of region I in catalysis and aphidicolin interaction are proposed. The mutational studies presented in this report will serve as a prototype for the functional role of region I in catalysis for all alpha-like DNA polymerases.

    View details for Web of Science ID A1993LD46600049

    View details for PubMedID 8496164

  • INITIATION OF SIMIAN VIRUS-40 DNA-REPLICATION REQUIRES THE INTERACTION OF A SPECIFIC DOMAIN OF HUMAN DNA POLYMERASE-ALPHA WITH LARGE T-ANTIGEN MOLECULAR AND CELLULAR BIOLOGY Dornreiter, I., Copeland, W. C., Wang, T. S. 1993; 13 (2): 809-820

    Abstract

    Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.

    View details for Web of Science ID A1993KH79300010

    View details for PubMedID 8380896

  • CELL-CYCLE EXPRESSION OF 2 REPLICATIVE DNA-POLYMERASES ALPHA AND DELTA FROM SCHIZOSACCHAROMYCES-POMBE MOLECULAR BIOLOGY OF THE CELL Park, H., Francesconi, S., Wang, T. S. 1993; 4 (2): 145-157

    Abstract

    We have investigated the expression of two Schizosaccharomyces pombe replicative DNA polymerases alpha and delta during the cell cycle. The pol alpha+ and pol delta+ genes encoding DNA polymerases alpha and delta were isolated from S. pombe. Both pol alpha+ and pol delta+ genes are single copy genes in haploid cells and are essential for cell viability. In contrast to Saccharomyces cerevisiae homologs, the steady-state transcripts of both S. pombe pol alpha+ and pol delta+ genes were present throughout the cell cycle. Sequence analysis of the pol alpha+ and pol delta+ genes did not reveal the Mlu I motifs in their upstream sequences that are involved in cell cycle-dependent transcription of S. cerevisiae DNA synthesis genes as well as the S. pombe cdc22+ gene at the G1/S boundary. However, five near-match Mlu I motifs were found in the upstream region of the pol alpha+ gene. S. pombe DNA polymerases alpha and delta proteins were also expressed constantly throughout the cell cycle. In addition, the enzymatic activity of the S. pombe DNA polymerase alpha measured by in vitro assay was detected at all stages of the cell cycle. Thus, these S. pombe replicative DNA polymerases, like that of S. pombe cdc17+ gene, are expressed throughout the cell cycle at the transcriptional and protein level. These results indicate that S. pombe has at least two regulatory modes for the expression of genes involved in DNA replication and DNA precursor synthesis.

    View details for Web of Science ID A1993KQ09200002

    View details for PubMedID 8443413

  • HUMAN DNA POLYMERASE-ALPHA AND POLYMERASE-BETA ARE ABLE TO INCORPORATE ANTI-HIV DEOXYNUCLEOTIDES INTO DNA JOURNAL OF BIOLOGICAL CHEMISTRY Copeland, W. C., Chen, M. S., Wang, T. S. 1992; 267 (30): 21459-21464

    Abstract

    Deoxynucleoside analogs, AZT and/or ddN, are the therapeutic agents currently utilized to inhibit the human immunodeficiency virus (HIV) reverse transcriptase. The effects of their anabolic products, AZT-triphosphate (AZT-TP) and ddCTP on human cellular DNA metabolic processes were studied using highly purified, structurally and enzymatically defined forms of the two major human host DNA polymerases, alpha and beta, and compared to those of the reverse transcriptase purified from HIV viron. Human DNA polymerase alpha during processive DNA synthesis is able to incorporate AZT-monophosphate (AZT-MP) but not ddCMP into DNA, causing chain termination. During its initial encounter with a primer terminus, polymerase alpha is able to incorporate both AZT-MP and ddCMP into DNA chains. Polymerase beta is able to incorporate AZT-MP and ddCMP into DNA, causing chain termination in both modes of DNA synthesis. Steady state kinetic analyses demonstrate that polymerase alpha inserts one AZT-MP molecule into DNA for every 2500 dTMP molecules incorporated. Polymerase beta incorporates ddCMP with efficiency nearly equal to that of dCMP. HIV reverse transcriptase prefers to incorporate AZT-MP and ddCMP rather than dTMP and dCMP, respectively. The findings described here raise the concern that the capability of the two major host DNA polymerases to incorporate AZT-MP or ddCMP into DNA might cause adverse side effects on human DNA metabolism and mutation in the genomes of patients under long term continuous treatment with AZT and ddC.

    View details for Web of Science ID A1992JV01100032

    View details for PubMedID 1400458

  • PROTEIN AFFINITY-CHROMATOGRAPHY REVEALS CELL-CYCLE DEPENDENT ASSOCIATION OF CELLULAR FACTORS WITH HUMAN DNA POLYMERASE-ALPHA CHROMOSOMA Rogge, L., Wang, T. S. 1992; 102 (1): S114-S120

    Abstract

    DNA polymerase alpha/primase (Pol alpha) is the key replication enzyme in eukaryotic cells. This enzyme synthesizes and elongates short RNA primers at an unwound origin of replication. Pol alpha was used as an affinity ligand to identify cellular replication factors interacting with it. Protein complexes between Pol alpha and cellular factors were analyzed by co-immunoprecipitations with monoclonal antibodies directed against Pol alpha and by protein affinity chromatography of cell extracts derived from pure G1- and S-phase cell populations on Pol alpha affinity columns. Co-immunoprecipitations resulted in the identification of a polypeptide with a molecular weight of 46 kDa. For Pol alpha affinity chromatography, the ligand was purified from insect cells infected with a recombinant baculovirus encoding the catalytic subunit (p180) of Pol alpha (Copeland and Wang, 1991). With 5 x 10(8) infected Sf9 cells, a rapid one step purification protocol was used which yielded in five hours 0.6 mg pure enzyme with a specific activity of 140,000 units/mg. The G1- and S-phase cell populations were generated by block, release and counterflow centrifugal elutriation of exponentially growing human MANCA cells. Starting with 2 x 10(9) non synchronous cells, 5 x 10(8) G1-phase cells were isolated. Chromatography of cell extracts derived from G1- or S-phase cells on Pol alpha affinity columns resulted in identifying several polypeptides in the range of 40-70 kDa. Some of these polypeptides are more abundant in eluates derived from S-phase extracts than from G1-phase extracts.

    View details for Web of Science ID A1992KF85400016

    View details for PubMedID 1291232

  • CATALYTIC SUBUNIT OF HUMAN DNA POLYMERASE-ALPHA OVERPRODUCED FROM BACULOVIRUS-INFECTED INSECT CELLS - STRUCTURAL AND ENZYMOLOGICAL CHARACTERIZATION JOURNAL OF BIOLOGICAL CHEMISTRY Copeland, W. C., Wang, T. S. 1991; 266 (33): 22739-22748

    Abstract

    The human DNA polymerase alpha catalytic polypeptide has been functionally overexpressed by a recombinant baculovirus in insect cells at greater than 1000-fold higher levels than that found in cultured normal human cells. The recombinant polymerase alpha protein is translated from its natural translation start codon under the control of the baculovirus polyhedron promoter producing a protein of 180 kDa, identical in size to that isolated from cultured human cells. This recombinant polymerase alpha is phosphorylated and reactive to a panel of monoclonal antibodies directed against the native polymerase alpha-primase complex and to polyclonal antisera against N- and C-terminal peptides of the polymerase alpha catalytic polypeptide. The recombinant enzyme was immunopurified from insect cells as a single polypeptide. The single subunit recombinant polymerase alpha has no detectable 3'-5' exonuclease activity. The Km for primer-template and dNTP, reactivity to inhibitors, N2-(p-n-butylphenyl)-dGTP (BuPdGTP) and aphidicolin, thermosensitivity, and DNA synthetic processivity and fidelity of the recombinant polymerase alpha are identical to that observed with the four-subunit polymerase alpha-primase complex immunopurified from cultured human cells. These results strongly suggest that the presence of the other subunits, (the p70 and the two primase subunits, p48 and p58), does not influence kinetic parameters of polymerase alpha catalysis, sensitivity to inhibitors, or DNA synthetic fidelity and processivity.

    View details for Web of Science ID A1991GR56400100

    View details for PubMedID 1939281

  • CELL CYCLE-DEPENDENT PHOSPHORYLATION OF HUMAN DNA POLYMERASE-ALPHA JOURNAL OF BIOLOGICAL CHEMISTRY Nasheuer, H. P., Moore, A., Wahl, A. F., Wang, T. S. 1991; 266 (12): 7893-7903

    Abstract

    The expression of DNA polymerase alpha, a principal chromosome replication enzyme, is constitutive during the cell cycle. We show in this report that DNA polymerase alpha catalytic polypeptide p180 is phosphorylated throughout the cell cycle and is hyperphosphorylated in G2/M phase. The p70 subunit is phosphorylated only in G2/M phase. This cell cycle-dependent phosphorylation is due to cell cycle-dependent kinase(s) and not to phosphatase(s). In vitro evidence indicates the involvement of p34cdc2 kinase in the mitotic phosphorylation of DNA polymerase alpha. Tryptic phosphopeptide maps demonstrate that peptides phosphorylated in vitro are identical to those phosphorylated in vivo. DNA polymerase alpha from mitotic cells is found to have lower affinity for single-stranded DNA than does polymerase alpha from G1/S phase cells. These results imply that the mitotic phosphorylation of polymerase alpha may affect its physical interaction with other replicative proteins and/or with DNA at the replication fork.

    View details for Web of Science ID A1991FJ34200090

    View details for PubMedID 1902230

  • HUMAN DNA POLYMERASE-ALPHA GENE - SEQUENCES CONTROLLING EXPRESSION IN CYCLING AND SERUM-STIMULATED CELLS MOLECULAR AND CELLULAR BIOLOGY Pearson, B. E., Nasheuer, H. P., Wang, T. S. 1991; 11 (4): 2081-2095

    Abstract

    We have investigated the DNA polymerase alpha promoter sequence requirements for the expression of a heterologous gene in actively cycling cells and following serum addition to serum-deprived cells. An 11.4-kb genomic clone that spans the 5' end of this gene and includes 1.62 kb of sequence upstream from the translation start site was isolated. The transcription start site was mapped at 46 +/- 1 nucleotides upstream from the translation start site. The upstream sequence is GC rich and lacks a TATA sequence but has a CCAAT sequence on the opposite strand. Analysis of a set of deletion constructs in transient transfection assays demonstrated that efficient expression of the reporter in cycling cells requires 248 bp of sequence upstream from the cap site. Clustered within these 248 nucleotides are sequences similar to consensus sequences for Sp1-, Ap1-, Ap2-, and E2F-binding sites. The CCAAT sequence and the potential E2F- and Ap1-binding sites are shown to be protected from DNase I digestion by partially purified nuclear proteins. The DNA polymerase alpha promoter can confer upon the reporter an appropriate, late response to serum addition. No single sequence element could be shown to confer serum inducibility. Rather, multiple sequence elements appear to mediate the full serum response.

    View details for Web of Science ID A1991FC85200038

    View details for PubMedID 2005899

  • EUKARYOTIC DNA-POLYMERASES ANNUAL REVIEW OF BIOCHEMISTRY Wang, T. S. 1991; 60: 513-552

    View details for Web of Science ID A1991FV90900016

    View details for PubMedID 1883203

  • HUMAN DNA POLYMERASE-ALPHA CATALYTIC POLYPEPTIDE BINDS CONA AND RCA AND CONTAINS A SPECIFIC LABILE SITE IN THE N-TERMINUS NUCLEIC ACIDS RESEARCH Hsi, K. L., Copeland, W. C., Wang, T. S. 1990; 18 (21): 6231-6237

    Abstract

    The catalytic polypeptide of DNA polymerase alpha is often observed in vitro as a family of phosphopolypeptides predominantly of 180 and 165 kDa derived from a single primary structure. The estimated Mr of this polypeptide deduced from the full-length cDNA is 165 kDa. Immunoblot analysis with polyclonal antibodies against peptides of the N- and C-termini of the deduced primary sequence indicates that the observed family of polypeptides from 180 kDa to lower molecular weight results from proteolytic cleavage from the N-terminus. Antibodies against the N-terminal peptide detect only the 180 kDa species suggesting that this higher molecular weight polypeptide may be the result of posttranslational modification of the 165 kDa primary translation product. The catalytic polypeptide is not only phosphorylated but is also found to react with lectins ConA and RCA. N-terminal sequencing of the isolated catalytic polypeptide from human cells and of the recombinant fusion proteins indicates that the often observed 165 kDa polypeptide is the in vitro proteolytic cleavage product of the modified 180 kDa protein at the specific site between lys123 and lys124 within the sequence -RNVKKLAVTKPNN-.

    View details for Web of Science ID A1990EJ01100006

    View details for PubMedID 2243771

  • DNA POLYMERASES-ALPHA AND POLYMERASES-DELTA ARE IMMUNOLOGICALLY AND STRUCTURALLY DISTINCT JOURNAL OF BIOLOGICAL CHEMISTRY Wong, S. W., Syvaoja, J., Tan, C. K., Downey, K. M., So, A. G., Linn, S., Wang, T. S. 1989; 264 (10): 5924-5928

    Abstract

    The relationship between DNA polymerases alpha and delta are evaluated immunologically by monoclonal antibody specifically against DNA polymerase alpha and murine polyclonal antiserum against calf thymus DNA polymerase delta. DNA polymerases alpha and delta are found to be immunologically distinct. The structural relationship between the proliferating cell nuclear antigen (PCNA)-dependent calf DNA polymerase delta and DNA polymerase alpha from human and calf was analyzed by two-dimensional tryptic peptide mapping of the catalytic polypeptides. The results demonstrate that the catalytic polypeptides of the PCNA-dependent calf polymerase delta and DNA polymerase alpha are distinct, unrelated, and do not share any common structural determinants. The immunological and structural relationship between a recently identified PCNA-independent form of DNA polymerase delta from HeLa cells was also assessed. This PCNA-independent human polymerase delta was found to be immunologically unrelated to human polymerase alpha but to share some immunological and structural determinants with the PCNA-dependent calf thymus polymerase delta.

    View details for Web of Science ID A1989T999200095

    View details for PubMedID 2466832

  • HUMAN DNA POLYMERASE-ALPHA - PREDICTED FUNCTIONAL DOMAINS AND RELATIONSHIPS WITH VIRAL-DNA POLYMERASES FASEB JOURNAL Wang, T. S., Wong, S. W., Korn, D. 1989; 3 (1): 14-21

    Abstract

    The primary sequence of human DNA polymerase alpha deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage phi 19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

    View details for Web of Science ID A1989R828000004

    View details for PubMedID 2642867

  • GENE-EXPRESSION OF HUMAN DNA POLYMERASE-ALPHA DURING CELL-PROLIFERATION AND THE CELL-CYCLE MOLECULAR AND CELLULAR BIOLOGY Wahl, A. F., GEIS, A. M., Spain, B. H., Wong, S. W., Korn, D., Wang, T. S. 1988; 8 (11): 5016-5025

    Abstract

    We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

    View details for Web of Science ID A1988Q686900049

    View details for PubMedID 3211134

  • THE EVOLUTIONARY CONSERVATION OF DNA POLYMERASE-ALPHA NUCLEIC ACIDS RESEARCH Miller, M. A., Korn, D., Wang, T. S. 1988; 16 (16): 7961-7973

    Abstract

    The evolutionary conservation of DNA polymerase alpha was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase alpha monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. The most highly cross-reactive antibody recognizes a unique epitope on a 165-180 kDa catalytic polypeptide in cell lysates from several eukaryotic sources, as distant from man as the amphibians. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase alpha catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA polymerase alpha among vertebrates and the conservation of specific genetic sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.

    View details for Web of Science ID A1988P964900016

    View details for PubMedID 2458565

  • HUMAN DNA-POLYMERASE ALPHA-GENE EXPRESSION IS CELL-PROLIFERATION DEPENDENT AND ITS PRIMARY STRUCTURE IS SIMILAR TO BOTH PROKARYOTIC AND EUKARYOTIC REPLICATIVE DNA-POLYMERASES EMBO JOURNAL Wong, S. W., Wahl, A. F., Yuan, P. M., Arai, N., Pearson, B. E., Arai, K., Korn, D., Hunkapiller, M. W., Wang, T. S. 1988; 7 (1): 37-47

    Abstract

    We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.

    View details for Web of Science ID A1988L734900006

    View details for PubMedID 3359994

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