ENZYMOLOGY OF REPLICATION OF YEAST CHROMOSOMAL DNA

Summary

Principal Investigator: PETER BURGERS
Affiliation: Washington University School of Medicine
Country: USA
Abstract: The general goal of the proposed research is to describe the molecular architecture of the eukaryotic DNA replication fork, its function during normal replication, and its response to stress or DNA damage, using S. cerevisiae as a model system. Recent studies have delineated the activities of DNA polymerase d and DNA polymerase e at the standard replication fork, and the role of DNA polymerase d in lagging strand DNA replication. Translesion synthesis (TLS) in response to DNA damage allows the cell to overcome damage that forms a terminal block for the regular replication machinery; TLS is associated with an increased frequency of mutations, hence mutagenesis. A ubiquitinated form of the replication clamp PCNA initiates its essential roles in TLS through recruitment of the Rev1 protein, a factor that organizes the TLS machinery. The DNA damage checkpoint is a signal transduction pathway that temporarily holds the cell cycle in response to DNA damage. The checkpoint clamp 9-1-1, when brought together onto DNA with the sensor kinase Mec1 (human ATR), activates Mec1 to phosphorylate downstream targets including Rad53 (human Chk1/2). Preliminary studies indicate that the replication initiation protein kinase Cdc7-Dbf4 uniquely phosphorylates critical factors in the mutagenesis and checkpoint pathways. These data suggest that the three pathways are more interconnected than they at first appear to be. The proposal will test specific hypotheses central to genome replication and maintenance. In aim 1, the mechanistic and structural nature of strand displacement synthesis by DNA polymerase d, and its regulation by the flap endonuclease FEN1 will be investigated. In aim 2, the efficiency and fidelity of TLS by DNA polymerase ? and associated factors, and the role of the Cdc7-Dbf4 protein kinase in TLS and mutagenesis will be studied. In aim 3, the role of the checkpoint clamp 9-1-1 in checkpoint function and in mutagenesis will be addressed by biochemical studies and mutational analysis. The regulation of the 9-1-1 pathway by Cdc7-Dbf4 phosphorylation will also be studied. Proper replication of cellular DNA, and responses to stress and DNA damage is of the highest importance in maintaining the integrity of our genetic information. Patients with known defects in these pathways are at a highly increased risk for developing cancer. These pathways are conserved from human to yeast. The proposed studies will be carried out in yeast because this model organism is more approachable to genetic and biochemical analysis. PUBLIC HEALTH RELEVANCE: Proper replication of cellular DNA is of the highest importance in maintaining the integrity of our genetic information. In this application, we are proposing to study the process of DNA replication, and the responses of the cell to DNA damage that can lead to the generation of mutations and to cell death. Patients with known defects in these pathways are at a highly increased risk for developing cancer. These pathways are conserved from human to yeast, and we are proposing to study these pathways in yeast, because this model organism is more approachable to genetic and biochemical analysis.
Funding Period: 1984-04-01 - 2013-08-31
more information: NIH RePORT

Top Publications

  1. ncbi The multiple biological roles of the 3'-->5' exonuclease of Saccharomyces cerevisiae DNA polymerase delta require switching between the polymerase and exonuclease domains
    Yong Hwan Jin
    National Institute of Environmental Health Sciences, D3 01, 101 TW Alexander Dr, P O Box 12233, Research Triangle Park, NC 27709, USA
    Mol Cell Biol 25:461-71. 2005
  2. ncbi The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA
    Olga Chilkova
    Department of Medical Biochemistry and Biophysics, Department of Biochemistry, Umea University, 901 87 Umea, Sweden
    Nucleic Acids Res 35:6588-97. 2007
  3. ncbi Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya
    Josephine Wardle
    Institute for Cell and Molecular Biosciences ICaMB, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK
    Nucleic Acids Res 36:705-11. 2008
  4. ncbi Division of labor at the eukaryotic replication fork
    Stephanie A Nick McElhinny
    Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA
    Mol Cell 30:137-44. 2008
  5. ncbi Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae
    Debbie Sommer
    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198 6805, USA
    Nucleic Acids Res 36:4699-707. 2008
  6. ncbi Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal
    Marie L Rossi
    Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
    J Biol Chem 283:27483-93. 2008
  7. ncbi Dividing the workload at a eukaryotic replication fork
    Thomas A Kunkel
    Laboratory of Molecular Genetics and Laboratory of Structural Biology, 111 T W Alexander Drive, National Institute of Environmental Health Sciences, National Institute of Health, DHHS, Research Triangle Park, NC 27709, USA
    Trends Cell Biol 18:521-7. 2008
  8. ncbi Polymerase dynamics at the eukaryotic DNA replication fork
    Peter M J Burgers
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    J Biol Chem 284:4041-5. 2009
  9. ncbi Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase
    Vasundhara M Navadgi-Patil
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    J Biol Chem 283:35853-9. 2008
  10. ncbi Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis
    Carrie M Stith
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    J Biol Chem 283:34129-40. 2008

Scientific Experts

  • T A Kunkel
  • PETER BURGERS
  • Parie Garg
  • Carrie M Stith
  • Jerzy Majka
  • Vasundhara M Navadgi-Patil
  • Scott D McCulloch
  • Stephanie A Nick McElhinny
  • Göran O Bylund
  • Jason E Pike
  • Dmitry A Gordenin
  • Adam Wood
  • Hyongi Chon
  • Jana E Stone
  • Xuan Li
  • Robert A Bambara
  • Judith L Campbell
  • Debbie Sommer
  • Josephine Wardle
  • Marie L Rossi
  • Erik Johansson
  • Grace E Kissling
  • Olga Chilkova
  • Michael A Resnick
  • John M Fortune
  • Zhihao Zhuang
  • Matthew R Northam
  • Alexa A Franco
  • Yong Hwan Jin
  • Robert J Kokoska
  • Igor B Rogozin
  • Alex Vassilev
  • Susana M Cerritelli
  • Melvin L DePamphilis
  • Robert J Crouch
  • Yingming Zhao
  • Wolf Dietrich Heyer
  • Junmei Zhang
  • Scott A Lujan
  • Peter McGlynn
  • Wensheng Wang
  • Li-Jung Lin
  • Bernard A Connolly
  • Isaac K O Cann
  • Li Jung Lin
  • Kate Darley
  • Robert S Lahue
  • Jonathan Sanvoisin
  • Joan Sterling
  • Pauline Heslop
  • Peter Stenlund
  • Else Britt Lundström
  • Pawel Grabowski
  • Isabelle Isoz
  • Stephen J Benkovic
  • Bonita L Yoder
  • Anita Niedziela-Majka
  • Polina V Shcherbakova
  • Dmitri M Baitin
  • Marc S Wold
  • Sara K Binz
  • Joan F Sterling
  • Wendy M Lam
  • Paul D Kaufman
  • Laura J W Murray
  • Hanan Al-Refai
  • Carrie M W Stith

Detail Information

Publications34

  1. ncbi The multiple biological roles of the 3'-->5' exonuclease of Saccharomyces cerevisiae DNA polymerase delta require switching between the polymerase and exonuclease domains
    Yong Hwan Jin
    National Institute of Environmental Health Sciences, D3 01, 101 TW Alexander Dr, P O Box 12233, Research Triangle Park, NC 27709, USA
    Mol Cell Biol 25:461-71. 2005
    ..We conclude that the three biological functions of the 3'-->5' exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme...
  2. ncbi The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA
    Olga Chilkova
    Department of Medical Biochemistry and Biophysics, Department of Biochemistry, Umea University, 901 87 Umea, Sweden
    Nucleic Acids Res 35:6588-97. 2007
    ..We conclude that Pol epsilon and Pol delta exhibit comparable processivity, but are loaded on the primer-end via different mechanisms...
  3. ncbi Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya
    Josephine Wardle
    Institute for Cell and Molecular Biosciences ICaMB, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK
    Nucleic Acids Res 36:705-11. 2008
    ..The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil...
  4. ncbi Division of labor at the eukaryotic replication fork
    Stephanie A Nick McElhinny
    Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA
    Mol Cell 30:137-44. 2008
    ....
  5. ncbi Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae
    Debbie Sommer
    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198 6805, USA
    Nucleic Acids Res 36:4699-707. 2008
    ..Although additional LLR factors remain to be identified, the excision and resynthesis steps of LLR from a 5' nick can be reconstituted in a purified system with FEN1 and Pol delta, together with PCNA and its loader RFC...
  6. ncbi Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal
    Marie L Rossi
    Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
    J Biol Chem 283:27483-93. 2008
    ..Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal...
  7. ncbi Dividing the workload at a eukaryotic replication fork
    Thomas A Kunkel
    Laboratory of Molecular Genetics and Laboratory of Structural Biology, 111 T W Alexander Drive, National Institute of Environmental Health Sciences, National Institute of Health, DHHS, Research Triangle Park, NC 27709, USA
    Trends Cell Biol 18:521-7. 2008
    ..We also review earlier studies in light of this model and then consider prospects for future investigations of possible variations on this simple division of labor...
  8. ncbi Polymerase dynamics at the eukaryotic DNA replication fork
    Peter M J Burgers
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    J Biol Chem 284:4041-5. 2009
    ..In the more common short flap pathway, Pol delta coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase...
  9. ncbi Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase
    Vasundhara M Navadgi-Patil
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    J Biol Chem 283:35853-9. 2008
    ..Our studies suggest that Dpb11 and 9-1-1 may partially compensate for each other during yeast checkpoint function...
  10. ncbi Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis
    Carrie M Stith
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    J Biol Chem 283:34129-40. 2008
    ..Finally, RNA-DNA hybrids are more readily displaced by Pol delta than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation...
  11. ncbi Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex
    Hyongi Chon
    Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda MD 20892, USA
    Nucleic Acids Res 37:96-110. 2009
    ..Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype...
  12. ncbi The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases delta and eta
    Scott D McCulloch
    Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences Research, NC 27709, USA
    Nucleic Acids Res 37:2830-40. 2009
    ..The fact that yeast and mammalian Pol eta have intrinsically different catalytic properties has potential biological implications...
  13. ncbi Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase zeta
    Jana E Stone
    Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences Research, NIH, DHHS, Research Triangle Park, NC 27709, USA
    Nucleic Acids Res 37:3774-87. 2009
    ..This may explain the origin of some multiple clustered mutations observed in vivo...
  14. ncbi A tale of two tails: activation of DNA damage checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1
    Vasundhara M Navadgi-Patil
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, United States
    DNA Repair (Amst) 8:996-1003. 2009
    ..cerevisiae Dpb11 or by vertebrate TopBP1: activation is mediated by the intrinsically disordered C-terminal tail of each activator. The relative contributions made by multiple activators of Mec1/ATR are discussed...
  15. ncbi Pif1 helicase lengthens some Okazaki fragment flaps necessitating Dna2 nuclease/helicase action in the two-nuclease processing pathway
    Jason E Pike
    Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
    J Biol Chem 284:25170-80. 2009
    ..However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1...
  16. ncbi PCNA is required for initiation of recombination-associated DNA synthesis by DNA polymerase delta
    Xuan Li
    Department of Microbiology, University of California, Davis, 95616 8665, USA
    Mol Cell 36:704-13. 2009
    ..We conclude that PCNA has a specific role in the initiation of recombination-associated DNA synthesis and that DNA polymerase delta promotes recombination-associated DNA synthesis...
  17. ncbi Effects of accessory proteins on the bypass of a cis-syn thymine-thymine dimer by Saccharomyces cerevisiae DNA polymerase eta
    Scott D McCulloch
    Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA
    Biochemistry 46:8888-96. 2007
    ..Thus, although accessory proteins clearly participate in pol eta functions in vivo, they do not appear to help suppress UV mutagenesis by improving pol eta bypass fidelity per se...
  18. ncbi A ubiquitin-binding motif in the translesion DNA polymerase Rev1 mediates its essential functional interaction with ubiquitinated proliferating cell nuclear antigen in response to DNA damage
    Adam Wood
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA
    J Biol Chem 282:20256-63. 2007
    ..Taken together, these studies suggest a specific mechanism by which the interaction between Rev1 and ubiquitinated PCNA is stabilized during the DNA damage response...
  19. ncbi How the cell deals with DNA nicks
    Parie Garg
    Department of Biochemistry and Molecular Physics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
    Cell Cycle 4:221-4. 2005
    ..cerevisiae Pol delta. These same parameters are also important for other DNA metabolic processes, such as base excision repair, that depend on Pol delta for synthesis...
  20. ncbi DNA polymerases that propagate the eukaryotic DNA replication fork
    Parie Garg
    Washington University School of Medicine, St. Louis, MO 63110, USA
    Crit Rev Biochem Mol Biol 40:115-28. 2005
    ..The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute...
  21. ncbi Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase zeta
    Parie Garg
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
    J Biol Chem 280:23446-50. 2005
    ..However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta...
  22. ncbi Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C
    Alexa A Franco
    Lawrence Berkeley National Laboratory and Department of Molecular and Cell Biology, University of California, Berkeley, 94720, USA
    Genes Dev 19:1365-75. 2005
    ..We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery...
  23. ncbi Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex
    Göran O Bylund
    Department of Biochemistry, Washington University School of Medicine, 660 S Euclid, St Louis, Missouri 63110, USA
    Mol Cell Biol 25:5445-55. 2005
    ....
  24. ncbi Function of Rad17/Mec3/Ddc1 and its partial complexes in the DNA damage checkpoint
    Jerzy Majka
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA
    DNA Repair (Amst) 4:1189-94. 2005
    ..In agreement, overexpression of DDC1 or RAD17 in a MEC3Delta strain, or of MEC3 or RAD17 in a DDC1Delta strain shows no rescue of damage sensitivity...
  25. ncbi Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1
    Parie Garg
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110, USA
    Proc Natl Acad Sci U S A 102:18361-6. 2005
    ..We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication...
  26. ncbi The structure of a ring-opened proliferating cell nuclear antigen-replication factor C complex revealed by fluorescence energy transfer
    Zhihao Zhuang
    Department of Chemistry, 414 Wartik Laboratory, Pennsylvania State University, University Park, PA 16802, USA
    Proc Natl Acad Sci U S A 103:2546-51. 2006
    ..The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm...
  27. ncbi Overproduction and purification of RFC-related clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae
    Göran O Bylund
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri, USA
    Methods Enzymol 409:1-11. 2006
    ..This methodology yielded all clamp loaders in high yield and with high enzymatic activity. The yeast 9-1-1 checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, was overproduced and purified in a similar manner...
  28. ncbi Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions
    Jerzy Majka
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
    J Biol Chem 281:27855-61. 2006
    ..These studies show a unique specificity of the checkpoint loader for 5'-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed...
  29. ncbi RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase delta
    John M Fortune
    Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA
    Nucleic Acids Res 34:4335-41. 2006
    ..Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences...
  30. ncbi A novel function of DNA polymerase zeta regulated by PCNA
    Matthew R Northam
    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
    EMBO J 25:4316-25. 2006
    ....
  31. ncbi Inefficient proofreading and biased error rates during inaccurate DNA synthesis by a mutant derivative of Saccharomyces cerevisiae DNA polymerase delta
    Stephanie A Nick McElhinny
    Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA
    J Biol Chem 282:2324-32. 2007
    ..cerevisiae strain has an elevated spontaneous mutation rate that is likely due to reduced replication fidelity in vivo...
  32. ncbi The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint
    Jerzy Majka
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    Mol Cell 24:891-901. 2006
    ..Phosphorylation and binding studies with individual clamp subunits indicate that the Ddc1 subunit mediates the functional interactions with Mec1...
  33. ncbi Clamping the Mec1/ATR checkpoint kinase into action
    Jerzy Majka
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA
    Cell Cycle 6:1157-60. 2007
    ..Both mechanisms of activation generally upregulate the kinase activity towards all downstream targets...
  34. ncbi The unstructured C-terminal tail of the 9-1-1 clamp subunit Ddc1 activates Mec1/ATR via two distinct mechanisms
    Vasundhara M Navadgi-Patil
    Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA
    Mol Cell 36:743-53. 2009
    ..Remarkably, small peptides that fuse the two tryptophan-containing motifs together are proficient in activating Mec1...