The long range goals of the proposed research are: 1) to elucidate the molecular mechanisms of DNA double-strand break (DSB) induced recombination between homologous genes in various chromosomal environments, and 2) to investigate the relationships between genetic recombination, transcription, and DNA repair. Recombination processes are involved in a variety of important biological 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 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 studies will utilize the yeast Saccharomyces cerevisiae HO nuclease to introduce DSBs at defined loci within or near repeated genes and centromere DNA, and in transcriptionally active and silent genes. The fate of DSBs in different chromosomal contexts will be monitored by using four complementary approaches, including measurements of recombination frequencies, genetic and physical analyses of recombinant products, and kinetic analyses of DSB induction and repair. Specific questions to be addressed include the following: 1. How does the relative position of a DSB to duplicated DNA affect the genetic consequences of DSB-repair? 2. What are the lengths and origins of gene conversion tracts induced by DSBs? 3. What are the properties and consequences of DSB-induced recombination near and within centromeres? 4. What effect does transcription have on the formation of heteroduplex DNA, and on the repair of mismatches within heteroduplex DNA? 5. What are the roles of specific DNA-repair gene products in spontaneous and DSB-induced recombination, and in the correction of mismatches in heteroduplex DNA? These studies will clarify the mechanisms and genetic consequences of spontaneous and DSB-induced recombination in a variety of chromosomal environments, further our understanding of the roles of specific DNA repair gene products in these processes, and provide a basis for understanding the relationships between DNA damage repair, transcription, and recombination.
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