The role of replication fidelity in mutator phenotypes and carcinogenesis.

The overall god of this dissertation is to investigate the role of specific biochemical determinants of DNA replication fidelity in genomic stability and tumor progression in mammals. This dissertation also describes the development of an assay to examine the effects of potential mutator genes on spontaneous mutation frequency in vivo . Chapter 2 describes an examination of the roles of specific alleles of the 3 ' -5 ' exonuclease of yeast polymerase 8 in determining spontaneous mutation. Alteration of two conserved amino acids implicated in exonucleolytic catalysis caused potent mutator phenotypes, showing that these residues are required for genomic stability. This is the first analysis of these residues in a eukaryotic nuclear polymerase. Yeast homologues of human polymerase ? variants in mutator cell lines caused a modest increase in spontaneous mutation rate. The interaction of these proofreading mutants with a defect in mismatch repair resulted in synergistic mutator effects. These findings imply that there may be multiple components to mutator phenotypes in cancer. The generation of a mutator phenotype could be an early step in carcinogenesis. Chapter 3 describes a test of this hypothesis employing a mutant hamster CTP synthetase gene, previously shown to cause a mutator phenotype in Chinese hamster ovary cells. To examine effects on tumorigenesis in vivo , this mutant was expressed as a transgene in mice. Five independent transgenic mouse lines showed significantly increased tumor incidence in multiple tissues. These results imply that the mutator CTP synthetase gene is sufficient for carcinogenesis in mammals. The measurement of mutation frequencies in vivo is important to understand the role of changes in mutation rate in disease. Chapter 4 of this dissertation describes an assay to measure the frequency of base substitution mutations using transgenic bacteriophage-derived shuttle vectors as mutagenesis targets in mice. Recovery of the transgenes by polymerase chain reaction and quantitation of mutation frequency using a genetic test in E. coli showed that this strategy successfully recovered sufficient numbers of transgenes, but it posed limitations on the sensitivity of the assay. These indings indicate that alternate strategies may be required to measure rare mutations occurring in mammalian cells.