Our goal is to understand the mechanism of meiotic recombination in mammals, and to determine how this process is monitored by the cell. In S. cerevisiae, recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. We recently cloned and disrupted mouse Spo11. Our results strongly suggest that the mechanism of recombination initiation is highly conserved. We propose to continue using the Spo11 knockout mouse to study meiotic recombination and the cellular responses to recombination defects.
The Specific Aims are as follows: 1. Checkpoint responses to recombination defects. We have proposed that mammals have sexually dimorphic DNA damage-dependent and -independent meiotic checkpoints. To test this idea, we will determine the epistasis relationships between early (Spo11-/-), middle (Dmc1-/- and Msh5-/-), and late (Mlh1-/-) recombination mutants, which elicit distinct checkpoint responses. We will also determine if p53 plays a role in oocyte apoptosis caused by recombination defects. 2. Putative DSB repair proteins and their role in mammalian meiotic recombination. Many proteins form foci on meiotic chromosomes and thus are assumed to be involved in repairing meiotic DSBs. We will determine the genetic requirements for formation of these foci by analyzing Spo11-/- and other mutants. In addition, we have developed a system to generate a site-specific DSB in spermatocytes using the rare-cutting endonuclease I-Scel. We will determine how such a break is repaired during wild-type meiosis and will introduce this system into a Spo11-/- background to monitor the assembly of putative recombination protein foci on a defined DSB site. 3. Spo11 function and cellular responses to partial recombination defects. We will express Spo11 transgenes under the control of a spermatocyte-specific promoter to determine the functional significance of known Spo11 splice variants and will express catalytically inactive mutants to determine if Spo11 has a DSB-independent role(s) during meiosis. We will also use these transgenes to titrate Spo11 activity in vivo. This will allow us to determine how cells respond to a modest recombination defect, as opposed to the catastrophic defects in recombination-defective null mutants. 4. Interplay between meiotic recombination and the oocyte spindle. Chiasma formation is thought to be critical for proper assembly of the first meiotic spindle in mouse oocytes. We will further explore this relationship by examining spindle formation in cultured Spo11-/- oocytes. We will also explicitly test whether Spo11 is required for meiotic crossing over by assaying chiasma formation in mutant oocytes.
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