The goals of the proposed research are to determine the mechanisms of double-strand break (DSB)-induced recombinational repair and associated mismatch repair in various chromosomal environments in wild-type, radiation-sensitive, and mismatch repair-defective strains of the yeast Saccharomyces cerevisiae. Recombination is a fundamental biological process involved in a variety of important phenomena, such as the regulation of gene expression, antigenic variation, repair of DNA DSBs, chromosomal translocations, and in mammals, antibody gene assembly. There is strong evidence linking genetic recombination and defects in mismatch repair to the development of a wide range of cancers in humans. DNA damage, such as that produced by ionizing radiation or chemical agents, stimulates recombination which may result in gene restoration, or produce mutagenic/carcinogenic alterations. Both DNA repair processes and homologous recombination are influenced by transcriptional activity. Although the effects of transcription on these processes are not well understood, they may play important roles in determining the genetic consequences of DNA repair. For example, differential processing of DNA damage in actively transcribed and silent regions of a genome may account for the diverse responses of various cell types, or of cells in different developmental states, to particular DNA damaging agents. The proposed research employs HO nuclease to cleave unique genomic sites in yeast, a controlled system for modeling DNA damage-induced recombination by agents such as radiation, chemicals, or endogenous nucleases. Both physical and genetic analyses are proposed to define the mechanisms, genetic consequences and genetic control of DSB-induced recombinational repair, and to clarify the influences of chromosomal environment on recombinational repair and mismatch repair. The three Specific Aims will address several questions, including: * What are the lengths and structures of spontaneous and DSB-induced gene conversion tracts, and how are these affected by allele linkage and the length of shared homology? * Is DSB-induced gene conversion a consequence of mismatch repair, gap repair, or both? * By what mechanism(s) does transcription influence the length of conversion tracts (i.e., by increasing lengths of heteroduplex DNA tracts or by stimulating mismatch repair)? * What genetic controls regulate DSB-induced reciprocal and nonreciprocal recombination? By answering these questions, we will clarify the mechanisms and genetic consequences of spontaneous and DSB-induced recombination. We will further our understanding of the influence of transcription on recombinational repair and on mismatch repair processes, clarify how these processes are regulated and gain insight into the genetic consequences of their misregulation. Ultimately, these studies will provide a basis for understanding the relationships among four DNA dynamic processes: DNA damage repair, transcription, recombination, and mismatch repair.
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