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

• 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
• 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' 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

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.

在减数分裂的预言I期间，诱导数百个双链断裂（DSB）启动同源 重组（HR），这可能导致形成跨界（CO），这对于维持 染色体的相互作用一直通过直到并确保在第一个减数分裂分裂中进行准确的隔离。仅有的 10％的DSB注定会成为COS，其他DSB被修复为非交叉（NCO），但所有DSB 必须及时修复，以防止基因组损害。两个不同类别的cos可以 发生：I类主要机械，涉及DNA不匹配维修（MMR）途径的组件，A 由MUS81-EME1核酸内切酶驱动的小型II类途径。选择的机制 这些CO途径或NCO途径的发生尚不清楚，尽管该途径的位置和频率 最终二线必须严格和精确地调节，以确保在第一个减数分裂时准确隔离 分配。在小鼠中，法科尼贫血（FA）相关蛋白SLX4对于指导CO事件很重要 走向两个主要途径之一；缺乏SLX4的小鼠向I类COS进行了转变，失去了II类 cos和持续的未经修复的DSB在预言I的末尾，表型与缺乏小鼠相似的表型 MUS81。这表明SLX4可以调节II类CO事件。 SLX4与大量DNA相互作用 修复因子，包括几个结构特定的内切酶（SSE； XPF-ERCC1，MUS81-EME1，SLX1）， 以及FA和MMR途径的组件。在鼠标中，我们证明了它与 SLX1对于DSB修复并不重要，而是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解旋酶的功能相互作用，该酶调节CO/NCO决策 在晚期I中，通过双霍利迪交界处（DHJ）修复中间体的解散。我们也有 在FA途径Fancj中确定了与另一个解旋酶的新型相互作用。电流的目标 建议是阐明在预言I期间驱动不同DSB修复途径的作用SLX4，以及 我们假设该角色取决于其与维修网络中主要参与者的互动。在AIM 1中， 我们将探索BLM，SLX4和MUS81之间的遗传相互作用，特别询问SLX4是否为 运作于编排II类事件，或者是否需要促进DHJ的BLM介导溶解。 在AIM 2中，我们将使用优雅的鼠标确定涉及SLX4的关键功能相互作用 模型系统地询问SLX4的每个相互作用合作伙伴。在AIM 3中，我们将探索角色和 使用我们的突变小鼠在预言I期间FANCJ和SLX4在减数分裂重组中的共依赖性 结合蛋白质组学分析的模型，以了解这两种蛋白质如何在功能上相互作用 在与之相互作用的不同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相互作用组的第一个功能分析 减数分裂，并将通过相交的修复途径来阐明如何实现全基因组CO。