The goals of the proposed research are to clarify the mechanisms, genetic consequences, and interrelationships of recombination and mismatch repair in the model eukaryote, Saccharomyces cerevisiae. Additional goals are to clarify the genetic control of spontaneous and double-strand break (DSB)-induced recombination, and the effects of transcription on mismatch repair, recombination, and mutagenesis. In many of the proposed studies, HO nuclease will be used to cleave unique, defined genomic sites in yeast chromosomes; this is a controlled system for modeling DNA damage-induced recombination by genotoxic agents such as radiation. DNA repair and genetic recombination are ubiquitous and fundamental cellular processes that are involved, for example, in gene regulation, immune system development, and genetic instability associated with cellular transformation and tumor progression. Recombination is stimulated by many agents that damage DNA, such as ionizing radiation and radiomimetic chemicals; these agents are also known to be mutagenic and carcinogenic. Mismatch repair defects are common in many forms of cancer.
Five Aims are proposed.
Aim 1 focuses on the role of mismatch repair during DSB-induced gene conversion. We will determine whether allelic gene conversion is mediated by mismatch repair; whether conversion on opposite sides of a DSB is mediated by independent mismatch repair tracts; and whether one or more heterozygosities increase conversion tract lengths.
Aim 2 focuses on the repair of loop mismatches. Loop mismatch repair varies among organisms, it differs from single-base mismatch repair, and it is influenced by loop structure. We will determine whether unpaired bases in a stem-loop influence repair efficiency, and the efficiency of nonpalindromic loop repair.
Aim 3 focuses on donor choice during DSB-induced gene conversion, including the effects of donor proximity and transcription levels, and the minimum length of a donor locus required for efficient DSB-induced gene conversion.
Aim 4 focuses on the genetic control of spontaneous and DSB-induced recombination. We will employ both knock-out and overexpression strategies to investigate the roles of XRS2, RAD51, and RAD52 in recombinational repair. An overexpression strategy will also be used to identify new genes involved in spontaneous and/or DSB- induced recombination.
Aim 5 focuses on transcriptional effects on DNA dynamics. We will determine whether transcription influences single-base mismatch repair, gene conversion tract directionality, allele conversion preference for spontaneous events; and mutagenic loss of palindromic sequences. These studies will provide insight into mechanistic aspects of DSB repair, transcription, recombination, and mismatch repair. These DNA dynamic processes are important in cancer etiology and cancer therapy (e.g., radiotherapy), and in technical aspects of gene therapy. Studies of transcriptional effects on DNA dynamics will provide insight into the different genetic consequences of DNA damage, repair, and recombination during development and during different stages of the cell cycle.
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