Eukaryotic cells possess multiple repair/tolerance pathways that deal with DNA damage in an """"""""error free"""""""" manner that maintains the integrity of the original DNA sequence. If damage escapes these pathways, however, it can be bypassed by an """"""""error prone"""""""" mechanism in which a low fidelity translesion polymerase inserts a nucleotide opposite a noncoding lesion. This latter type of pathway is mutagenic, and is responsible not only for virtually all mutations induced by environmental mutagens, but also for a substantial fraction of mutations that arise spontaneously. This project will use the yeast Saccharomyces cerevisiae as a model to examine the role of the Pol zetu translesion polymerase in both spontaneous and induced mutagenesis. Use will be made of a novel """"""""signature"""""""" for Pol zetu translesion synthesis activity that has been identified using a frameshift-specific mutation assay. This signature is comprised of complex mutations in which the selected frameshift mutation is accompanied by one or more nonselected base substitution mutations. The frameshift-specific assay will be used to further explore the inter-relationships between the Pol zetu translesion pathway and other DNA damage repair/tolerance pathways in yeast. Both spontaneous mutations as well as mutations induced by common environmental mutagens will be examined. In addition, the mechanism for the generation of Pol zetu-dependent complex mutations will be investigated by molecular manipulation of a defined hotspot for complex events. Finally, in order to determine whether multiple base substitutions are a general feature of Pol zetu translesion synthesis, an assay will be developed that can specifically detect multiple base substitutions that arise in the absence of a frameshift mutation. The proposed studies will elucidate basic mechanisms of spontaneous and induced mutagenesis, and so are relevant to the accumulation of multiple mutations during tumorigenesis.
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