Genetic analysis of early events in mammalian homologous recombination in vivo

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In eukaryotic organisms, homologous recombination (HR) serves a dual role. On one hand, it results in crossover formation between homologous chromosomes during meiosis, in effect, shuffling the genetic material of both parents and leading to the generation of new combinations of alleles and genetic traits. On the other hand, in mitotically-dividing cells, HR serves in the repair of DNA damage, especially DNA double strand breaks (DSB) and thus, faithfully promotes genome integrity. Several human cancers and genome-destabilizing diseases that confer increased susceptibility to cancer are linked to defects in HR. In eukaryotes, the central step in the repair of a DNA DSB involves the binding of multiple monomers of the RAD51 protein to 3' ending single stranded DNA created by nucleolytic resection. The resulting RAD51 nucleoprotein filament promotes formation of a joint molecule between the processed broken DNA and the homologous repair template by the orchestrated steps of homology searching, DNA strand invasion and exchange. Joint molecule formation is followed by new DNA synthesis, which replaces nucleotides lost through DSB formation and 3' end resection. Subsequent steps may involve unwinding (dissolution) of the DNA strand containing the newly synthesized DNA and ligation to the processed second end, or the formation of a stably-joined Holliday junction intermediate that can be processed further to yield recombinant products. Unfortunately, most of our knowledge of the HR process comes from studies in microbial systems. Our laboratory has developed sensitive assays to critically assess the mechanisms of HR in mammalian cells. As part of this effort, we developed an assay that measures the nascent DNA formed by polymerizing 3' ends in vivo. In recent work, we exploited this assay in the analysis of the homology requirements for 3' polymerization and the analysis of the role of Rad51 and Brca2 in the process. Thus, our laboratory is in the very unique position of having developed a very sensitive assay for studying the biochemical genetics of very early events that occur during the process of mammalian homologous recombination in vivo and we are requesting NSERC support for this research work. As detailed in the proposal, this application has 5 specific aims focused on the biochemical genetic analysis of the 3' polymerization process, namely, 1. Functional analysis of Rad52-epistasis group proteins and recombination mediators in the early events of HR. 2. Detecting Rad51:DNA interactions at a 3' invading end by chromatin immunoprecipitation (CHIP) analysis. 3. To conduct a physical analysis of the 3' polymerization process 4. Investigate the role of short tract DNA polymerases involved in 3' polymerization 5. Chromosome conformation capture (CCC) analysis.