This project will investigate effects of the genetic background of the cell on mechanisms of spontaneous and radiation-induced mutagenesis in human lymphoblast cells. Recently, a p53-null human lymphoblast line was created and it was shown that, unlike in certain p53-mutated lines, the cells are not hypermutable.
Specific Aim 1. The fact that p53-null cells are apparently not hypermutable, but are hyperrecombinational, has led to the hypothesis that spontaneous mutagenesis at endogenous alleles and recombination in integrated vectors are not equivalent indicators of genomic instability. To date, these two endpoints have not been compared in a single cell system, and that is the goal of Aim 1. A matched pair of vectors called pHWT and pHWD that can measure recombination at the bacterial gpt locus will be integrated into cells that are p53 wild type, p53-mutated and p53-null. Background mutation frequencies at two endogenous gene loci, X-linked hypoxanthine guanine phosphoribosyltransferase (hprt) and autosomal, heterozygous thymidine kinase (tk), will be compared with recombination at gpt.
Specific Aim 2. It is hypothesized that p53 involvement in double strand break (DSB) repair is responsible for its effects on mutagenesis, via involvement in homologous recombination (HR), non-homologous endjoining (NHEJ), or both.
Aim 2 is to study the genetics of mutagenesis, by determining the quantitative and qualitative effects of disrupting NHEJ or HR. The relatively new technique of chimeraplasty will be used to create targeted knockouts of specific genes involved in DSB repair, including KU8O and XRCC4 to disrupt NHEJ, and RAD51 and RAD54 to disrupt HR. When feasible, both """"""""pure"""""""" knockouts in which both alleles are inactivated and also """"""""conditional"""""""" knockouts where the wild type allele is added back under control of a regulated promoter will be created. For each new line, spontaneous and radiation-induced mutation frequencies will be determined at hprt and tk. Also, for each new cell line, and also for p53 wild type, p53-mutated, and p53-null, the radiation-induced mutational spectra for hprt and tk will be determined.