Structural and functional principles underlying germline genome transmission

种系基因组传播的结构和功能原理

• 批准号: 10535616
• 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
• 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/10535616
• 关键词: Address; Affect; Affinity; Aneuploidy; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Process; Biology; Cells; Child; Child Development; Chromosome Pairing; Chromosome Segregation; Chromosome abnormality; Collaborations; Collection; Complex; Computer Models; Computing Methodologies; Cryoelectron Microscopy; Crystallization; Cytology; DNA; DNA Binding; DNA Double Strand Break; 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; Lead; Location; Mammals; Maps; Meiosis; Meiotic Recombination; Methods; Modeling; Molecular; Molecular Genetics; Mouse Protein; Mus; Mutation; NMR Spectroscopy; Nature; Nucleoproteins; Parents; Phenotype; 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; egg; experimental study; homologous recombination; in vivo; innovation; novel strategies; offspring; protein structure; recruit; reproductive success; reproductive system disorder; single molecule; sperm cell; stem cells; stoichiometry; transmission process; yeast genetics

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育取决于准确的传播 遗传物质从父母传给孩子。减数分裂过程中的同源重组在这一过程中起着核心作用 通过确保准确的染色体分离实现遗传传播。重组中的错误可能会导致 配子的非整倍体或突变，进而导致儿童流产或发育缺陷。 因此，了解重组的机制和调控对于了解减数分裂如何进行至关重要。 错误影响人类生育和儿童发育，但重组的分子原理仍然存在 由于缺乏生化和结构信息，人们对此还不完全了解。减数分裂重组 由Spo11蛋白与一套 辅助因素。我们最近通过首次提纯克服了长期存在的进步障碍 DSB促进蛋白的重组复合体。在这一进展的基础上，Keeney和Patel实验室 建议扩展他们正在进行的合作，将生化、结构和单分子结合在一起 体外生物物理方法和体内功能实验，以阐明 控制Spo11形成DSB的方式。通过对小鼠的蛋白质进行平行研究 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有方面的能力。目标1将专注于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-EM， X射线结晶学，计算模型和生化研究，以确定结构 Spo11核心复合体及其关键的蛋白质-蛋白质和蛋白质-DNA界面。我们还将测试 我们在体内的结构和生化发现的生理学相关性。为此，我们将使用分子 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化遗传 竞争性移植转基因精原干细胞池在小鼠中的筛选 生殖细胞枯竭小鼠的睾丸。目标2将专注于保守的辅助蛋白Rec114、Mei4和 Mer2，它们是调节DSB的时间、数量和位置的重要纽带。我们将使用核磁共振 光谱学、X射线结晶学、低温电子显微镜和计算机建模来定义结构和蛋白质- 异三聚体Rec114-Mei4复合体和同四聚体Mer2复合体的蛋白质界面。我们会 使用整体生物化学和单分子生物物理方法来定义机制和动力学 在这些蛋白质协同组装以在DNA上形成核蛋白缩合物的背后，我们 假设是他们支持Spo11活动能力的一个核心特征。我们还将应用电池中的 活体试验是为了测试结构和生化发现所产生的功能预测。