Biochemistry of recombination in gametogenesis

描述（由申请人提供）：减数分裂是配子发生的核心部分，对有性生殖至关重要。在减数分裂中，染色体复制一次，然后分离两次，产生单倍体减数分裂产物。一个保守的减数分裂特异性同源重组途径确保了减数分裂I中染色体的正确分离。在配子发生过程中，重组的完全丧失会引发细胞凋亡，从而导致不育。因此，减数分裂重组是可逆的，受精前，男性避孕药的候选目标。减数分裂前期双链DNA（dsDNA）断裂引发减数分裂。体内dsDNA断裂的形成需要许多基因，但对其各自的蛋白质知之甚少。其中一种蛋白，Red 2（Spoil），与MB型拓扑异构酶的催化亚基是邻位的，并且涉及催化重组性dsDNA断裂的形成。虽然这一暗示是在大约十年前提出的，但尚未报道该蛋白质的体外活性。我们报告，红2，其推定的活性位点酪氨酸，和DNA结合基序是必不可少的重组。我们纯化了Rec12相关的复合物，其中包含六个已知的减数分裂重组所需的蛋白质和四个蛋白质与推断的生化活性的重组。我们还纯化了从两种不同来源（细菌、营养酵母细胞）表达的重组Red 2。减数分裂蛋白复合物和纯化的Red 2的每种制备物都可以在体外切割dsDNA。可以在体外区分特异性影响DNA结合或催化的氨基酸取代。这一时期的重点是红2和相关蛋白质的生物化学。具体目的是：（1）确定Red 2结合和切割DNA的生化机制。(2)确定Red 2在体内和体外功能所必需的关键残基。(3)确定来自减数分裂的Red 2蛋白复合物的组成。(4)开发用于高通量筛选潜在抗Red 2化合物的体外测定法。这些结果将揭示对人类生殖生物学缺陷具有潜在诊断价值的保守蛋白质。它们还将为合理的药物设计和高通量筛选铺平道路，以确定专门影响Rec12依赖性功能的潜在避孕药，从而触发减数分裂特异性细胞凋亡，而不会对体组织产生不良副作用。

DESCRIPTION (provided by applicant): 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.