Tolerance of spontaneous and induced DNA damage in yeast

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Genomic DMA is the target of a wide variety of endogenous and exogenous DMA-damaging agents. The resulting lesions are an important source of mutagenesis and genome instability, and contribute to genetic disease, cancer and aging in humans. To alleviate the deleterious effects of DNA damage, there are specialized excision mechanisms that remove lesions to restore the integrity of duplex DNA. In spite of the presence of these highly-efficient excision repair mechanisms, lesions can persist and be encountered during DNA synthesis. Some lesions alter the base-pairing properties of the affected nucleotide, leading to mutations during DNA synthesis, while others have the potential to block the progress of a replication fork. To counteract the presence of polymerase-blocking lesions, cells possess redundant damage tolerance mechanisms to promote the bypass of DNA damage, thereby allowing replication to continue. These bypass mechanisms include high-fidelity strand-switching and homologous recombination processes that copy information from an undamaged sister chromatid, as well as potentially error-prone pathways that involve the recruitment of specialized translesion synthesis (TLS) DNA polymerases. Because of the relative ease of genetic manipulation, the yeast Saccharomyces cerevisiae provides an excellent model system for studying these highly conserved DNA damage processing mechanisms. Aims 1 and 2 of this proposal will focus on the bypass/tolerance of spontaneous DNA damage, with an emphasis on defining the genetic control of the error-free versus error-prone bypass pathways. Aims 3 and 4 will expand these studies to examine the cell cycle-dependent consequences of lesions induced by a model mutagen, ultraviolet (UV) light. While most UV-induced mutations are assumed to occur during lesion bypass in S phase, we also will examine mutations that arise in the context of the excision repair process. Finally, Aim 5 will address whether, in addition to their lesion bypass activity, the TLS polymerases also are important for extending mismatches incorporated by replicative DNA polymerases. Together, these studies will advance our understanding of the damage-related mechanisms that contribute to eukaryotic genome instability, a process that is central in the development of human diseases.