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.
