Biochemistry of recombination in gametogenesis

配子发生重组的生物化学

基本信息

批准号：
7896253
负责人：
Wayne P Wahls
金额：
$ 24.5万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7896253
关键词：
Active Sites Adverse effects Affect Amino Acid Substitution Apoptosis Bacteria Binding Biochemical Biochemical Reaction Biochemistry Biological Biological Assay Biological Models Catalysis Catalytic Domain Cells Chromosome Segregation Chromosomes Cleaved cell Complex Congenital Abnormality Contraceptive Agents Contraceptive methods Coupled DNA DNA Binding Defect Diagnostic Diploid Cells Drug Design Ensure Eukaryota Fertilization Fission Yeast Funding Gametogenesis Genes Genetic Recombination Goals Haploidy Human In Vitro Infertility Male Contraceptive Agents Mass Spectrum Analysis Meiosis Meiotic Recombination Mental Retardation Methods Modeling Molecular Pathway interactions Precipitation Pregnancy loss Preparation Process Prophase Proteins Reaction Recombinants Reporting Reproduction Reproductive Biology Research Personnel SPO11 gene Site-Directed Mutagenesis Source Sterility Structure Systems Analysis Testing Tissues Topoisomerase Tyrosine Yeasts base contraceptive target design high throughput screening homologous recombination in vitro Assay in vitro activity in vivo innovation interest male new technology programs protein complex protein protein interaction reconstitution small molecule yeast two hybrid system

项目摘要

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.

描述（由申请人提供）：减数分裂是配子发生的中心部分，对于有性繁殖至关重要。在减数分裂中，染色体复制一次，然后隔离两次以产生单倍体减数分裂产物。保守的，减数分裂特异性的同源重组途径可确保减数分裂中染色体的适当分离I.重组的完全损失会触发配子发生过程中的凋亡，从而触发无菌。因此，减数分裂重组是可逆性，预先利用，男性避孕药的候选靶标。重组是由减数分裂预言中引起的双链DMA（DSDNA）断裂引发的。在体内形成DsDNA断裂所需的许多基因，但对它们各自的蛋白质知之甚少。其中一种蛋白质是红色2（变性），与MB型拓扑异构酶的催化亚基直源，与催化重组DSDNA断裂的形成有关。尽管这一含义是十年前大约产生的，但尚无蛋白质的体外活性。我们报告说，红色2，其推定的活性位点酪氨酸和DNA结合基序对于重组至关重要。我们纯化了一种与REC12相关的复合物，该复合物包含六种已知的减数分裂重组所需的蛋白质和四种具有重组的生化活性的蛋白质。我们还纯化了从两个不同来源（细菌，营养酵母细胞）表达的重组红2。减数分裂蛋白复合物和纯化的红色2的每种制剂都可以在体外裂解dsDNA。可以在体外区分影响特异性DNA结合或催化的氨基酸取代。这一时期的重点是红色2和相关蛋白的生物化学。具体目的是：（1）确定红色2与DNA结合并切割DNA的生化机制。（2）确定红色2的关键残基，对于体内和体外功能必不可少。（3）从减数分裂中确定红色2蛋白复合物的组成。（4）开发体外测定法，以对潜在抗红色2种化合物进行高吞吐量筛选。结果将揭示人类生殖生物学缺陷的潜在诊断价值的保守蛋白质。他们还将为理性的药物设计和高通量筛查铺平道路，以鉴定影响特别依赖REC12依赖功能的潜在避孕药，从而触发减数分裂特异性的细胞凋亡，而不会对体细胞组织产生不利的副作用。