The main goal of the research is to study the structure and function of DNA polymerase delta (Pol-delta), the principal DNA polymerase in the cell for DNA replication and for DNA repair. Studies will be carried out in the yeast Saccharomyces cerevisiae, an excellent model system for basic studies in DNA metabolism. Recent studies from this laboratory have shed new light on the structure of Pol-delta. Most significantly and intriguingly, the enzyme has an overall dimeric structure. The most obvious implication of these new findings is that Pol-delta may function as a dimer at the DNA replication fork. These recent studies have been facilitated by the availability of the genes for the two small subunits of Pol-delta, POL31 and POL32, and of overexpression systems in yeast and in E. coli. The proposed studies will focus on gaining an understanding of the functional significance of the dimeric structure of Pol-delta at the replication fork and the role of the small subunits of Pol-delta in establishing this structure. Major questions addressed in this proposal are: How do the dimerization domain and the Proliferating Cell Nuclear Antigen (PCNA)- binding domain of the Pol32p subunit affect yeast cell growth and damage-response? Recent studies indicate that dimerization and PCNA- binding reside in this subunit. Random and site-directed mutagenesis will be carried out to eliminated either function and test the consequences in vivo and in vitro. Is Pol-delta a functional dimer in vitro? Specific substrates will be designed to test this question. These studies will be facilitated by the availability of monomeric Pol-delta mutants. Which strand(s) does Pol-delta replicate and what is the role of Pol-epsilon? Studies will be carried out in permeable cells with specific nucleotide analogs. Does Pol-delta have additional subunit(s)? A suppressor analysis of strains mutant in POL31 or POL32 will be carried out to isolate a putative additional subunit. These studies are important for a further understanding of the mechanism of DNA replication in the eukaryotic cell. As some DNA replication factors also function in DNA repair, an understanding of the regulation and the interaction of these two pathways in yeast is important for understanding analogous processes in humans. Improper regulation of these processes in humans may lead to cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM058534-01
Application #
2729078
Study Section
Biochemistry Study Section (BIO)
Project Start
1999-01-01
Project End
2002-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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McCulloch, Scott D; Kokoska, Robert J; Chilkova, Olga et al. (2004) Enzymatic switching for efficient and accurate translesion DNA replication. Nucleic Acids Res 32:4665-75
Majka, Jerzy; Burgers, Peter M J (2004) The PCNA-RFC families of DNA clamps and clamp loaders. Prog Nucleic Acid Res Mol Biol 78:227-60
Ayyagari, Rao; Gomes, Xavier V; Gordenin, Dmitry A et al. (2003) Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2. J Biol Chem 278:1618-25
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Kamath-Loeb, A S; Loeb, L A; Johansson, E et al. (2001) Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence. J Biol Chem 276:16439-46
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