The principle aim of this proposal is to gain an understanding of the molecular mechanisms of translesion replication in eukaryotes. This process promotes DNA chain elongation past sites of template damage, at which normal replication is stalled, and is chiefly significant because it is a major source of induced and spontaneous mutations. Information about this process is therefore likely to provide important insights into carcinogenesis and genotoxicity in humans. Much of the proposed work will employ the technically amenable model eukaryote, the budding yeast Saccharomyces cerevisiae.
Specific aims i nclude the isolation of new genes whose products are involved in translesion replication, by virtue of their association with the Rev1, Rev3, or Rev7 proteins; isolation of the REV6 and NGM2 genes, thought to be involved in translesion replication; and identification of other genes with related function, by fractioning cell extracts for proteins that enhance translesion replication by DNA polymerase zeta and Rev1 protein in vitro.
Other aims i nclude in vivo analysis of REV or related gene functions by transforming deletion strains with plasmids carrying site specific UV photoproducts or abasic sites; enzymatic studies of the gene products, particularly in conjunction with pol zeta and Rev1 protein; and a mutational analysis of REV gene function. Information from yeast will be used to identify the human homologs of yeast genes, and to investigate the enzymatic properties of human DNA polymerase zeta, human Rev1 protein, and other gene products identified that have a related function. A secondary aim of the proposal is to investigate the mechanism of the RAD6-dependent error-free DNA damage tolerance pathway in yeast, which diminishes mutagenesis by unrepaired DNA damage. For this purpose, excision defective mutant strains that carry rad5, rad6, rad18, and rad30 deletions, or the pol30-46 allele (all of which are believed to involved in this pathway), will be transformed with double-stranded vectors that carry mutagenic lesions in both strands at staggered, variable positions. These plasmids have proved useful in detecting different kinds of recombinant events, a likely mechanism for the pathway.
