Biochemistry of recombination in gametogenesis

描述(申请人提供)：减数分裂是配子发生的中心部分，对有性繁殖是必不可少的。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。一条保守的、减数分裂特异的同源重组途径确保了减数分裂I中染色体的适当分离。完全失去重组会在配子发生过程中触发细胞凋亡，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靶标。重组是由减数分裂前期诱导的双链DNA(DsDNA)断裂启动的。体内dsDNA断裂的形成需要许多基因，但对它们各自的蛋白质知之甚少。其中一种蛋白质Red 2(SPOIL)与MB型拓扑异构酶的催化亚单位同源，与催化形成重组双链DNA断裂有关。虽然这一暗示是在大约十年前提出的，但该蛋白的体外活性尚未见报道。我们报告说，红色2，其假定的活性部位酪氨酸，和一个DNA结合基序是重组所必需的。我们纯化了一个与Rec12相关的复合体，它包含6个已知的减数分裂重组所需的蛋白质和4个推测具有重组生化活性的蛋白质。我们还纯化了来自两个不同来源(细菌、营养酵母细胞)的重组Red 2。该减数分裂蛋白复合体和每一种纯化的红2制剂都能在体外切割dsDNA。在体外可以区分影响DNA结合或催化的氨基酸取代。这一时期的重点是红色2和相关蛋白质的生物化学。其具体目的是：(1)确定Red 2与DNA结合和切割的生化机制。(2)确定体内和体外发挥功能所必需的Red 2关键残基。(3)确定一个来自减数分裂的红色2蛋白复合体的组成。(4)建立高通量筛选潜在抗红2类化合物的体外检测方法。这些结果将揭示保守蛋白质对人类生殖生物学缺陷的潜在诊断价值。他们还将为合理的药物设计和高通量筛选铺平道路，以确定潜在的避孕药，这些避孕药专门影响Rec12依赖的功能，从而触发特定于减数分裂的细胞凋亡，而不会对身体组织产生不良影响。

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.