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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取决于基因的准确传播