Enzymology of Replication of Yeast Chromosomal DNA

Washington University, Saint Louis MO

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Abstract

DESCRIPTION (provided by applicant): 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.