Genetics/DNA Precursor Metabolism, Mutagenesis, Repair

Gudas, Lorraine

Abstract

The major objective of this research is to employ genetic and biochemical techniques to elucidate the relationships between purine and pyrimidine metabolism and DNA repair, mutagenesis and carcinogenesis in mammalian cells. The second objective involves the use of somatic cell genetics to generate mutants with alterations in DNA repair pathways; characterization of these mutants will provide information concerning the biochemical regulation and the enzymology of DNA repair. We will utilize both a mouse S49 T-lymphoma cell line and a mouse teratocarcinoma stem cell line, F9, in these experiments. To accomplish these objectives, we first propose to further characterize mutants we've selected which exhibit alterations in either thymidylate synthetase of ribonucleotide reductase activity, in order to determine how these mutations influence DNA repair and mutagenesis. We will also perform mutagenesis experiments with these mutants which may provide evidence for an """"""""SOS"""""""" repair system in mammalian cells analogous to the """"""""SOS"""""""" system in E. coli. In addition, we will select a mutant totally deficient in ribonucleotide reductase activity in order to study mutagenesis in a line in which the deoxyribonucleotide levels can be more easily manipulated. Since deoxyribonucleosides can influence both the cytotoxicity and mutagenicity of alkylating agents, we plan to employ mutants such as the TTP feedback resistant ribonucleotide reductase mutant to further explore the mechanism by which nucleosides can greatly enhance the cytotoxicity of MNNG (N-methyl-N'-nitro-nitrosoguanidine) without enhancing the mutagenicity. In F9 teratocarcinoma cells we will investigate the mechanism by which partial thymidine starvation enhances the toxicity of retinoic acid, an agent which induces differentiation in these cells. Finally, we will select and characterize several new types of mutants including a uracil-DNA glycosylase deficient mutant, mutants resistant to specific types of alkylation (ie. a 1-methyldeoxyguanosine resistant mutant), and mutants with increased sensitivity to alkylating agents, or UV-light. These interrelated projects will help us understand how synthesis of DNA precursors and repair of DNA are related and how defects in these processes may result in mutagenesis, toxicity, or carcinogenesis.