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.

人类生殖的成功和健康后代的发育依赖于准确的传播减数分裂过程中从父母到孩子的遗传物质起着核心作用。通过确保准确的染色体分离来进行遗传传递可能会导致错误。配子的非整倍性或突变，进而导致流产或儿童发育缺陷。因此，了解重组的机制和调控对于理解减数分裂如何进行至关重要。错误影响人类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减数分裂重组信息，尚不完全了解。 DNA 双链断裂 (DSB) 是由 Spo11 蛋白与一系列我们最近通过首次纯化克服了长期存在的进步障碍。基于这一进展，Keeney 和 Patel 实验室开发了 DSB 促进蛋白的重组复合物。建议扩大他们正在进行的合作，将生化、结构和单分子结合起来体外生物物理方法和体内功能实验阐明了分子原理通过对小鼠蛋白质进行平行研究，控制 Spo11 形成 DSB 的过程。和酿酒酵母，我们将深入研究进化保守过程的机制同时保留探索哺乳动物特定方面的能力，目标 1 将重点关注 Spo11 的“核心复合体”。与其直接结合伙伴 TOP6BL（哺乳动物）和 Rec102–Rec104–Ski8（酵母）我们将应用冷冻电镜， X 射线晶体学、计算模型以及生化研究来定义结构 Spo11 核心复合物及其关键的蛋白质-蛋白质和蛋白质-DNA 界面我们还将测试。我们的体内结构和生化发现的生理相关性为此，我们将使用分子生物学。酵母的遗传、基因组和细胞学研究，并将采用一种新的方法来并行遗传通过竞争性地将转基因精原干细胞库移植到小鼠中进行筛选生殖细胞耗尽小鼠的测试将重点关注保守的辅助 Rec 蛋白114、Mei4 和 Mer2，作为调节 DSB 时间、数量和位置的纽带，我们将使用 NMR。光谱学、X射线晶体学、冷冻电镜和计算模型来定义结构和蛋白质- 我们将研究异源三聚体 Rec114-Mei4 复合物和同源四聚体 Mer2 复合物的蛋白质界面。使用大量生化和单分子生物物理方法来定义机制和动力学这些蛋白质协同组装在 DNA 上形成核蛋白凝聚物的背后，我们我们还将应用电池体内测定来测试由结构和生化发现产生的功能预测。