Biochemistry of recombination in gametogenesis

配子发生重组的生物化学

基本信息

批准号：
7302820
负责人：
Wayne P Wahls
金额：
$ 27.38万
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：
2007
资助国家：
美国
起止时间：
2007-08-10 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7302820
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 Eukaryotic Cell 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 Thinking Tissues Topoisomerase Tyrosine Yeasts base contraceptive target design high throughput screening homologous recombination human SPO11 protein in vitro Assay in vivo innovation interest male new technology programs 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核心部分，对于有性生殖至关重要。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。保守的、减数分裂特异性的同源重组途径确保了减数分裂 I 中染色体的正确分离。重组的完全丧失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目标。重组是由减数分裂前期诱导的双链 DMA (dsDNA) 断裂引发的。体内双链 DNA 断裂的形成需要许多基因，但人们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基是直系同源的，并且与催化重组双链 DNA 断裂的形成有关。尽管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 Red 2、其假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 Rec12 相关复合物，其中包含已知减数分裂重组所需的六种蛋白质和四种具有推断重组生化活性的蛋白质。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的每种制剂都可以在体外切割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 Red 2 和相关蛋白质的生物化学。具体目标是： (1) 确定 Red 2 结合和切割 DNA 的生化机制。 (2) 鉴定Red 2体内外功能所必需的关键残基。 (3)确定减数分裂中Red 2蛋白复合物的组成。 (4) 开发高通量筛选潜在抗 Red 2 化合物的体外测定方法。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。它们还将为合理的药物设计和高通量筛选铺平道路，以识别特异性影响 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。