The main goal of the research is to gain an understanding 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 experimental system. This proposal focuses on DNA polymerase 8 (Pol 8), the principal DNA polymerase in the cell for DNA replication and for DNA repair, and on that of the replication clamp PCNA and the related clamp Radl7/Mec3/Ddcl, whose functions are elicited in response to DNA damage. PCNA is the organizing center of the fork; it not only stabilizes the DNA polymerases at the fork, but also acts as an assembly factor for a large number of other factors involved in DNA metabolism, and interacts functionally with cell cycle control and damage response factors to regulate the structure and activity of the fork. The proposed studies are designed to further increase our understanding of the lagging strand replication machinery and the DNA damage response machineries.
In aim 1, the domains on the individual subunits of Pol delta that mediate interactions with PCNA will be mapped, and subjected to mutational analysis, in order to understand the contribution of each of these interactions during lagging strand DNA replication, translesion synthesis and mutagenesis.
In aim 2, the function of PCNA and of an ubiquitinated form of PCNA in translesion synthesis by Pol delta and by other translesion DNA polymerases will be investigated.
In aim 3, the function of the PCNA-like circular clamp Radl7/ Mec3/Ddcl and of the Mecl ATR-like kinase in establishing a temporary halt of the cell cycle, in response to DNA damage, will be the focus of study. Because components of the replication machinery and the damage response machinery are conserved in eukaryotes, we expect that an understanding of the mechanism and regulation of these machineries in yeast will serve as a model for understanding these processes in human cells. Improper function and regulation of these processes in humans may lead to the accumulation of mutations and chromosome abnormalities, and in cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032431-24
Application #
7452193
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Portnoy, Matthew
Project Start
1984-04-01
Project End
2009-08-31
Budget Start
2008-07-01
Budget End
2009-08-31
Support Year
24
Fiscal Year
2008
Total Cost
$499,107
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Burgers, Peter M J; Kunkel, Thomas A (2017) Eukaryotic DNA Replication Fork. Annu Rev Biochem 86:417-438
Koc, Katrina N; Singh, Saurabh P; Stodola, Joseph L et al. (2016) Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase ?. Nucleic Acids Res 44:3811-9
Burgers, Peter M J; Gordenin, Dmitry; Kunkel, Thomas A (2016) Who Is Leading the Replication Fork, Pol ? or Pol ?? Mol Cell 61:492-493
Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M (2016) Proficient Replication of the Yeast Genome by a Viral DNA Polymerase. J Biol Chem 291:11698-705
Stojkovi?, Gorazd; Makarova, Alena V; Wanrooij, Paulina H et al. (2016) Oxidative DNA damage stalls the human mitochondrial replisome. Sci Rep 6:28942
Stodola, Joseph L; Burgers, Peter M (2016) Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale. Nat Struct Mol Biol 23:402-8
Kochenova, Olga V; Bezalel-Buch, Rachel; Tran, Phong et al. (2016) Yeast DNA polymerase ? maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations. Nucleic Acids Res :
Cho, Jang-Eun; Huang, Shar-Yin N; Burgers, Peter M et al. (2016) Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo. Nucleic Acids Res 44:7714-21
Wanrooij, Paulina H; Burgers, Peter M (2015) Yet another job for Dna2: Checkpoint activation. DNA Repair (Amst) 32:17-23
Sparks, Justin L; Burgers, Peter M (2015) Error-free and mutagenic processing of topoisomerase 1-provoked damage at genomic ribonucleotides. EMBO J 34:1259-69

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