Structural and functional principles underlying germline genome transmission

种系基因组传播的结构和功能原理

基本信息

批准号：
10676300
负责人：
Scott Keeney
金额：
$ 48.9万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-03 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10676300
关键词：
Affect Affinity Aneuploidy Binding Biochemical Biological Assay Biological Process Biology Cells Child Child Development Chromosome Pairing Chromosome Segregation Collaborations Collection Complex Computer Models Computing Methodologies Cryoelectron Microscopy Cytology DNA DNA Binding DNA Double Strand Break DNA mapping Defect Development Developmental Disabilities Engraftment Ensure Fertility Genetic Genetic Materials Genetic Recombination Genetic Screening Genetic study Genome Genomics Germ Cells Goals Human In Vitro Infertility Joints Location Mammals Meiosis Meiotic Recombination Methods Modeling Molecular Molecular Genetics Mouse Protein Mus Mutation NMR Spectroscopy Nature Nucleoproteins Parents Phenotype Physical condensation Physiological Play Process Property Proteins Publications Recombinants Regulation Reproductive Health Resolution Role SPO11 gene Saccharomyces cerevisiae Site-Directed Mutagenesis Spo11 protein Spontaneous abortion Structure Surface Testing Testis Time Topoisomerase Transplantation X-Ray Crystallography Yeasts biophysical analysis biophysical techniques chromosome number abnormality egg experimental study homologous recombination in vivo innovation novel strategies offspring parallelization protein structure recruit reproductive development reproductive success reproductive system disorder single molecule sperm cell stem cells stoichiometry transmission process yeast genetics

项目摘要

Human reproductive success and the development of healthy offspring depend on accurate transmission of genetic material from parent to child. Homologous recombination during meiosis plays a central role in this genetic transmission by ensuring accurate chromosome segregation. Errors in recombination can lead to aneuploidy or mutations in gametes that in turn cause miscarriage or developmental defects in children. Understanding the mechanism and regulation of recombination is thus critical for understanding how meiotic errors affect human fertility and child development, but the molecular principles of recombination remain incompletely understood because of a paucity of biochemical and structural information. Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by the Spo11 protein in collaboration with a suite of accessory factors. We recently overcame longstanding barriers to progress by purifying for the first time recombinant complexes of DSB-promoting proteins. Building on this advance, the Keeney and Patel labs propose to extend their ongoing collaboration to combine biochemical, structural, and single molecule biophysical approaches in vitro with functional experiments in vivo to illuminate the molecular principles that govern how DSB formation by Spo11 occurs. By conducting these studies in parallel on proteins from mouse and Saccharomyces cerevisiae, we will dive deeply into the mechanisms of evolutionarily conserved processes while retaining the ability to explore mammal-specific aspects. Aim 1 will focus on a “core complex” of Spo11 with its direct binding partners TOP6BL (mammals) and Rec102–Rec104–Ski8 (yeast). We will apply cryo-EM, x-ray crystallography, and computational modeling along with biochemical studies to define the structure of Spo11 core complexes and their critical protein-protein and protein-DNA interfaces. We will also test the physiological relevance of our structural and biochemical findings in vivo. To this end, we will use molecular genetic, genomic, and cytological studies in yeast and will employ a novel approach to parallelized genetic screening in mouse by competitively transplanting pools of genetically modified spermatogonial stem cells into testes of germ cell-depleted mice. Aim 2 will focus on the conserved accessory proteins Rec114, Mei4, and Mer2, which are important as a nexus for regulating DSB timing, number and location. We will use NMR spectroscopy, x-ray crystallography, cryo-EM, and computational modeling to define the structures and protein- protein interfaces of heterotrimeric Rec114–Mei4 complexes and of homotetrameric Mer2 complexes. We will use bulk biochemical and single molecule biophysical approaches to define the mechanism and dynamics behind the cooperative assembly of these proteins to form nucleoprotein condensates on DNA, which we hypothesize to be a central feature of their ability to support Spo11 activity. We will also apply a battery of in vivo assays to test functional predictions arising from the structural and biochemical findings.

人类生殖成功和健康后代的发展取决于准确的传播从父母到孩子的遗传物质。减数分裂过程中的同源重组在此起着核心作用通过确保准确的染色体分离来传播遗传。重组中的错误可能导致在游戏中的非整倍性或突变会导致儿童流产或发育缺陷。因此，了解重组的机制和调节对于了解减数分裂的方式至关重要错误影响人类的生育能力和儿童发育，但重组的分子原理仍然存在由于缺乏生化和结构信息，因此不完全理解。减数分裂重组用SPO11蛋白与一套Spo11蛋白制造的DNA双链断裂（DSB）的启动附件因素。最近，我们首次净化了净化的长期障碍 DSB促蛋白的重组复合物。基尼和帕特尔实验室以此为基础提议扩展其持续合作以结合生化，结构和单分子通过体内功能实验在体外进行生物物理方法，以阐明分子原理控制SPO11的DSB形成方式。通过对小鼠的蛋白质并行进行这些研究和酿酒酵母的葡萄糖，我们将深入研究进化构成过程的机理同时保留探索哺乳动物特定方面的能力。 AIM 1将重点放在SPO11的“核心综合体”上其直接结合伙伴Top6BL（哺乳动物）和REC102 – REC104 -SKI8（酵母）。我们将应用Cryo-Em， X射线晶体学和计算建模以及生化研究，以定义 SPO11核心复合物及其临界蛋白质蛋白质和蛋白-DNA界面。我们还将测试我们在体内结构和生化发现的生理相关性。为此，我们将使用分子酵母中的遗传，基因组和细胞学研究，将采用一种新颖的方法来平行遗传通过竞争性地移植遗传修饰的精子干细胞筛选小鼠中的筛选细胞缺乏的小鼠的测试。 AIM 2将重点放在配置的配件蛋白REC114，MEI4和 MER2，对于调节DSB时序，数字和位置的联系很重要。我们将使用NMR 光谱，X射线晶体学，冷冻EM和计算建模，以定义结构和蛋白质 - 异三聚体REC114 – MEI4复合物和同型MER2复合物的蛋白质界面。我们将使用散装生化和单分子生物物理方法来定义机制和动力学在这些蛋白质的合作组装后面，以在DNA上形成核蛋白凝结物，我们假设是支持SPO11活动能力的主要特征。我们还将使用一电池体内测定测试由结构和生化发现引起的功能预测。