Meiosis, a central part of gametogenesis, is essential for sexual reproduction. In meiosis chromosomes replicate once, then segregate twice to produce haploid meiotic products. A conserved, meiosis-specific homologous recombination pathway ensures the proper segregation of chromosomes in meiosis I. Complete loss of recombination triggers apoptosis during gametogenesis and, hence, sterility. Thus, meiotic recombination is a candidate target for reversible, pre-fertilization, male contraceptives. Recombination is initiated by double-strand DMA (dsDNA) breaks induced in meiotic prophase. Many genes are required for formation of dsDNA breaks in vivo, but little is known about their respective proteins. One of the proteins, Red 2 (Spoil), is orthologous to the catalytic subunit of type MB topoisomerases and is implicated to catalyze formation of recombinogenic dsDNA breaks. Although this implication was made about ten years ago, no in vitro activities of the protein have been reported. We report that Red 2, its putative active site tyrosine, and a DNA binding motif are essential for recombination. We purified a Rec12-associated complex that contains six proteins known to be required for meiotic recombination and four proteins with inferred biochemical activities of recombination. We also purified recombinant Red 2 expressed from two different sources (bacteria, vegetative yeast cells). The meiotic protein complex and each preparation of purified Red 2 can cleave dsDNA in vitro. Amino acid substitutions affecting specifically DNA binding or catalysis can be distinguished in vitro. The focus for this period is upon the biochemistry of Red 2 and associated proteins.
The specific aims are: (1) To determine biochemical mechanisms by which Red 2 binds to and cleaves DNA. (2) To identify key residues of Red 2 essential for functions in vivo and in vitro. (3) To determine the composition of a Red 2 protein complex from meiosis. (4) To develop in vitro assays for high- throughput screening of potential anti-Red 2 compounds. The results will reveal conserved proteins of potential diagnostic value for defects in human reproductive biology. They will also pave the way for rational drug design and high-throughput screening to identify potential contraceptive agents that affect specifically Rec12-dependent function, thereby triggering meiosis-specific apoptosis without adverse side-effects on somatic tissues. ? ? ?
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