The long-term objective of this project is to understand the molecular mechanisms that are responsible for DNA-damage induced mutagenesis in bakers' yeast, Saccharomyces cerevisiae. Because yeast is a eukaryote, and moreover is unusually well developed experimentally, such knowledge of mutagenic mechanisms is likely to be of value for several health related concerns, such as cancer, genetic disease, and mutagenic hazard. In particular, it has become increasingly clear that the development of metastatic cancer frequently depends on the sequential occurrence of mutations in certain critical genes, and understanding this process may suggest preventive measures.
The specific aims for the proposed funding period are of two kinds. First, the capabilities of yeast cells for replicating DNA that contains a single defined lesion will be investigated. In addition to the frequency of such translesion synthesis, the error frequency and resulting mutation spectrum will also be determined. Comparisons between data for different lesions will be used to gain an insight into the reason why mutations occur. Lesions to be studied include cis-syn and trans-syn cyclobutane dimers, 6,4 pyrimidine pyrimidone dimers, and abasic sites. Effects of sequence context will be examined. Such work will use single- or double-stranded vectors that carry a single, uniquely located mutagenic lesion. Second, the molecular structure and function of genes and gene products that are concerned with mutagenesis will be investigated. Studies of REV3, one of these genes, that appears to encode a DNA polymerase used only for translesion synthesis, will in particular be studied. Similar studies will be carried out with other genes, such as REV7. In each case, the aim will be to determine the genes nucleotide sequence, to study its regulation, and purify its product. The eventual aim is to reconstruct mutagenic mechanisms in vitro, using purified proteins and templates that carry uniquely located lesions of different kinds.
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