Translesion synthesis by yeast DNA polymerases

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DESCRIPTION (provided by applicant): Our long-term objective is to delineate the mechanisms eukaryotic cells employ to overcome replication blockage caused by DNA lesions. The specific aims of this project are to elucidate the mechanisms of translesion synthesis (TLS) by DNA polymerases eta and n in the yeast Saccharomyces cerevisiae. In Aim 1, we will carry out biochemical and genetic studies to examine the hypothesis that the Rev1 DNA synthetic activity co-operates with Poleta_ to replicate past highly distorting DNA adducts originating from endogenous metabolism at the reactive N position of guanine. Aim 2 will test the idea that Rev1, via its interaction with PCNA, plays a crucial role in the targeting of Poleta to the lesion site. In Aim 3, proteins that are components of Poln, Revl, and Rev7 associated multi-protein assemblies will be purified, as will interacting proteins. Biochemical and genetic studies will define the roles of the newly identified protein factors in TLS. In Aim 4, in vivo and in vitro studies will examine the ubiquitination of Poln, Rev1, Rev3, Rev7, and other proteins, and ascertain the roles of the Rad6-Radl8 and Mms2-Ubcl3 ubiquitin conjugating enzyme complexes in these ubiquitination events. In Aim 5, the mechanism by which Poleta and Poln enter into the replication complex stalled at a lesion site will be determined. Our studies should yield important insights into the mechanisms of translesion synthesis by Poleta and Poln. The results will be highly relevant for cancer biology, as the manner in which DNA lesions are bypassed during replication has a major impact on genome stability, and, in fact, inactivation of Poln in humans leads to the cancer prone syndrome, the variant form of xeroderma pigmentosum.