SLX4 as a mediator of crossover pathway decisions in mammalian meiosis

SLX4 作为哺乳动物减数分裂中交叉途径决策的中介者

基本信息

批准号：
10540369
负责人：
Paula Elaine Cohen
金额：
$ 38.07万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-30 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10540369
关键词：
Animals Automobile Driving Binding Biochemical Cell Division Process Chromosome Pairing Chromosomes Codependence Congenital Abnormality Cruciform DNA DNA DNA Repair DNA Repair Pathway Data Double Strand Break Repair ERCC1 gene Ensure Event Exhibits Fanconi Anemia pathway Fanconi anemia protein Fanconi&apos s Anemia Fertilization Frequencies Genes Genetic Genetic Crossing Over Genome Germ Cells Goals Grant Human Individual Laboratories Lead Mammals Mediating Mediator Meiosis Meiotic Prophase I Meiotic Recombination Minor Mismatch Repair Molecular Mus Nature Normal Cell Outcome Pathway interactions Phenotype Play Ploidies Preparation Process Proteins Proteomics Regulation Resolution Role Saccharomycetales Scaffolding Protein Sexual Reproduction Somatic Cell Structure Topoisomerase III combinatorial endonuclease gene product genome-wide helicase homologous recombination member mouse model mutant mutant mouse model novel prenatal prevent recruit repaired segregation

项目摘要

The induction of hundreds of double strand breaks (DSB) during prophase I of meiosis initiates homologous recombination (HR), which can result in the formation of crossovers (CO) that are essential for maintaining chromosome interactions through until, and ensuring accurate segregation at, the first meiotic division. Only 10% of DSBs are destined to become COs, the others being repaired as non-crossovers (NCO), but all DSBs must be repaired in a timely and robust fashion to prevent genome damage. Two distinct classes of COs can occur: a major class I machinery, involving components of the DNA mismatch repair (MMR) pathway, and a minor class II pathway, driven by the MUS81-EME1 endonuclease. The mechanisms by which selection of these CO pathways, or NCO pathways, occurs remain unclear, although the placement and frequency of the final CO tally must be stringently and exquisitely regulated to ensure accurate segregation at the first meiotic division. In mouse, the Fanconi Anemia (FA) related protein, SLX4, is important for directing CO events towards one of the two major pathways; mice lacking Slx4 exhibit a shift towards class I COs, loss of class II COs, and persistent unrepaired DSBs at the end of prophase I, phenotypes similar to that of mice lacking Mus81. This indicates that SLX4 may regulate class II CO events. SLX4 interacts with a large number of DNA repair factors, including several structure specific endonucleases (SSEs; XPF-ERCC1, MUS81-EME1, SLX1), as well as components of the FA and MMR pathways. In mouse, we have demonstrated that its interaction with SLX1 is not critical for DSB repair, but that it interacts with the meiosis-specific MMR heterodimer, MutSγ. Moreover, our studies indicate a functional interaction with BLM helicase, which regulates CO/NCO decisions in late prophase I through the dissolution of double Holliday Junction (dHJ) repair intermediates. We have also identified a novel interaction with another helicase in the FA pathway, FANCJ. The goal of the current proposal is to elucidate the role SLX4 in driving different DSB repair pathways during prophase I, and we hypothesize that this role depends on its interaction with key players in the repair network. In Aim 1, we will explore the genetic interactions between BLM, SLX4, and MUS81, specifically asking whether SLX4 is functioning to orchestrate class II events, or whether it is required to promote BLM-mediate dissolution of dHJs. In Aim 2, we will identify key functional interactions involving SLX4 during prophase I, using elegant mouse models to systematically interrogate each interacting partner of SLX4. In Aim 3, we will explore the roles and co-dependence of FANCJ and SLX4 in meiotic recombination during prophase I, using our mutant mouse models, combined with proteomics analysis, to understand how these two proteins interact functionally to regulate CO/NCO decisions in the context of the different DNA repair pathways with which they interact. Collectively, these studies represent the first functional analysis of the SLX4 interactome in mammalian meiosis, and will illuminate how genome-wide CO is achieved through intersecting repair pathways.

减数分裂前期 I 期间诱导数百个双链断裂 (DSB) 启动同源重组（HR），这可能导致交叉（CO）的形成，这对于维持染色体相互作用直至第一次减数分裂，并确保精确分离。 10% 的 DSB 注定会成为 CO，其他的则作为非交叉 (NCO) 进行修复，但所有 DSB 必须及时、有效地修复，以防止基因组损伤有两种不同类型的 CO。发生：主要的 I 类机制，涉及 DNA 错配修复 (MMR) 途径的组成部分，以及由 MUS81-EME1 核酸内切酶驱动的次要 II 类途径选择的机制。尽管这些 CO 途径或 NCO 途径的发生位置和频率仍不清楚最终的 CO 计数必须受到严格而精细的控制，以确保第一次减数分裂时的准确分离在小鼠中，范可尼贫血 (FA) 相关蛋白 SLX4 对于指导 CO 事件非常重要。两条主要途径之一；缺乏 Slx4 的小鼠表现出向 I 类 CO 的转变，向 II 类的丧失 CO，以及前期 I 结束时持续未修复的 DSB，表型与缺乏的小鼠相似 Mus81。这表明SLX4可能调节II类CO事件，SLX4与大量DNA相互作用。修复因子，包括几种结构特异性核酸内切酶（SSE；XPF-ERCC1、MUS81-EME1、SLX1），以及 FA 和 MMR 通路的组成部分，我们已经在小鼠中证明了它与 FA 和 MMR 通路的相互作用。 SLX1 对于 DSB 修复并不重要，但它与减数分裂特异性 MMR 异二聚体 MutSγ 相互作用。此外，我们的研究表明与 BLM 解旋酶存在功能性相互作用，该解旋酶调节 CO/NCO 决策在前期 I 后期，我们还通过双霍利迪连接体 (dHJ) 的溶解修复中间体。确定了与 FA 途径中另一种解旋酶 FANCJ 的新型相互作用。提案旨在阐明 SLX4 在前期 I 期间驱动不同 DSB 修复途径的作用，以及我们认为这个角色取决于它与修复网络中关键参与者的互动。我们将探讨 BLM、SLX4 和 MUS81 之间的遗传相互作用，特别询问 SLX4 是否是协调 II 类事件的功能，或者是否需要促进 BLM 介导的 dHJ 解散。在目标 2 中，我们将使用优雅的鼠标来确定前期 I 期间 SLX4 的关键功能相互作用在目标 3 中，我们将探索 SLX4 的角色和角色。使用我们的突变小鼠，在前期 I 减数分裂重组中 FANCJ 和 SLX4 的相互依赖性模型与蛋白质组学分析相结合，以了解这两种蛋白质如何在功能上相互作用在它们相互作用的不同 DNA 修复途径的背景下调节 CO/NCO 决策。总的来说，这些研究代表了哺乳动物中 SLX4 相互作用组的首次功能分析。减数分裂，并将阐明如何通过交叉修复途径实现全基因组 CO。