UCLA

While much work has been done to elucidate the mechanisms through which mutations occur and the corresponding DNA repair, there is still much to be discovered in global pathways not directly related to the site of mutation, and how these pathways affect ensuing mutagenesis. In this dissertation, I will elucidate global pathways involved and how they influence both spontaneous and induced mutagenesis. We conducted a screen of six genotoxic agents against a single gene knockout library of Escherichia coli, and discovered a set of mutants that are involved in the cytotoxicity of these agents. In particular, folate biosynthetic pathways were found to be involved in the cell's resistance to a cytidine base analog. Next, using a papillation assay, we discovered four mutants involved in the nucleotide salvage pathway that show decreased levels of 5-azacytidine-induced mutagenesis, implicating RNA turnover and competing nucleotide pools in exacerbating this mutagenesis. We also show that the rNDP pool generated by the degradation of RNA is responsible for spontaneous mutations that result from replication errors, and these are normally repaired by the mismatch repair (MMR) system and prevented by 8-oxo-dGTP diphosphatase. Our results suggest that the rNDP pools derived from RNA degradation fuel replication to the point that replication errors escape the exonucleolytic editing function of DNA polymerase, but can be dealt with by the MMR system. We propose that in the absence of the cell's primary exoribonuclease (polynucleotide phosphorylase), the reduced rNDP pools limit replication to the point where the editing function can correct replication errors, and the MMR system is not required. Additionally, we discovered that in the absence of this exoribonuclease, there is an increase in frameshift mutations in the mutational spectrum, possibly due to the minimization of normal replication errors and revealing secondary mutations.