MAMMILIAN DOUBLE STRAND BREAK AND RECOMBINATIONAL REPAIR

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The goal of the proposed research is to test hypotheses about the mechanism of double-strand break (DSB)-induced homologous and non- homologous recombination (HR and NHR) in mammalian cells, including wild- type and mismatch repair-deficient cells, and in cells over-expressing recombinational repair proteins. Recombination is a fundamental biological process involved in many important phenomena, such as gene regulation, DSB repair, chromosomal translocations, and antibody gene assembly. There is strong evidence linking genetic recombination and defects in DNA repair to chancer. DNA damage stimulates recombination that is potentially mutagenic or carcinogenic. For technical reasons, most information about recombination in mammalian cells, particularly recombination induced by DNA damage, was derived from experiments with extrachromosomal DNA. Current evidence indicates that both HR and NHR differ markedly in extrachromosomal versus chromosomal DNA. This proposal concerns only the more biologically relevant chromosomal situation. The highly specific I-SceI nuclease from yeast will be used to cleave I- SceI sites within recombination substrates in vivo. Recombination substrates will be targeted to a specific mouse or human chromosomal locus to eliminate chromosome environment effects. This is a controlled system for modeling DNA damage-induced recombination by gents such as radiation, chemicals, or endogenous nucleases. Mechanistic studies relying on structural comparisons of substrate and product molecules are enhanced by using I-SceI nuclease since recombination initiation sites are under precise experimental control, which reduces the number of possible pathways from substrate to product and thus greatly simplifies the interpretation of results. Relating recombination substrates will be used to facilitate comparative analysis within and among individual projects. Both physical and genetic analyses will be performed to define the mechanisms, genetic consequences, and genetic control of DSB-induced recombinational repair. Specific Aim 1 addresses questions about DSB-induced HR mechanisms, with studies of gene conversion tract structure; association of gene conversion and crossing over; homology length and linkage requirements; and roles of mismatch repair. Specific Aim 2 concerns the relative rates of HR and NHR during chromosomal DSB repair, as well as mechanistic aspects such as the effects of homology interruptions on relative HR and NHR rates. Non- selective assays will provide a comprehensive view of the range of HR and NHR products that result from DSB repair. Specific Aim 3 involves the studies of the roles of mismatch and recombinational repair proteins during DSB-induced HR. These projects will greatly improve our understanding of in vivo DSB repair in mammalian chromosomes, and have relevance to the maintenance of genomic stability, particularly in cells exposed to DNA damaging agents that produce DSBs, such as ionizing radiation.