Meiosis and homologous recombination (HR) are critical functions in the reproduction, evolution, and biology of higher eukaryotes. HR is needed for proper chromosome segregation during meiosis, and repair of DNA damage in somatic cells. This proposal centers on genetic control of meiotic recombination, meiosis, and repair of DNA by HR in the mouse. Two basic strategies will be taken to identify and characterize genes involved in meiosis and HR. One is a gene-driven approach to analyze the function of the RecA homologs Dmc1 and Rad51d in meiosis. A mutation of the meiosis-specific Dmc1 gene causes meiotic arrest from chromosome synapsis failure. To better define the underlying defect, the null allele will be combined with transgenes and mutations that perturb checkpoint function, double strand break (DSB) induction, and an alternative HR pathway. These tests are designed to bypass the meiotic arrest, and obtain gametes for analysis of HR and chromosome segregation. Since a null mutation of Rad51d causes embryonic lethality, we will create a conditional knockout to evaluate this gene's role in meiotic HR. The other strategy for identifying meiosis and HR genes exploits a novel technology for whole genome mutagenesis using ES cells. One mutant we generated with this approach, mei1, causes sterility from meiotic prophase arrest. It exhibits defective chromosome synapsis, and failure to load Rad51 onto meiotic chromosomes. We will positionally clone mei1, study its gene product, and also characterize the defects in a second sterile mutation, mei2. Screens for additional meiotic mutants will be performed. Finally, a screen for DSB repair mutations will be conducted directly in mutagenized ES cells, enabling the derivation of mutant mice without the extreme husbandry requirements of traditional mutagenesis.
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