SLX4 as a mediator of crossover pathway decisions in mammalian meiosis

在减数分裂前期I诱导数百个双链断裂（DSB）， 重组（HR），这可能导致形成交叉（CO），这是维持 染色体相互作用，直到第一次减数分裂，并确保准确分离。只 10%的DSB注定要成为CO，其他的作为非交叉（NCO）进行修复，但所有DSB 必须以及时和稳健的方式进行修复，以防止基因组受损。两类不同的CO可以 发生：一种主要的I类机制，涉及DNA错配修复（MMR）途径的组分， 次要II类途径，由MUS 81-EME 1内切核酸酶驱动。选择的机制 这些CO途径，或NCO途径，发生仍然不清楚，虽然位置和频率的 最后的CO计数必须被严格和精细地调节，以确保在第一次减数分裂时的准确分离 师.在小鼠中，范可尼贫血（FA）相关蛋白SLX 4对于指导CO事件是重要的 缺乏Slx 4的小鼠表现出向I类CO的转变，II类CO的丧失， CO和在前期I结束时持续未修复的DSB，表型与缺乏 Mus81.这表明SLX 4可以调节II类CO事件。SLX 4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-ERCC 1、MUS 81-EME 1、SLX 1）， 以及FA和MMR途径的组分。在小鼠中，我们已经证明了它与 SLX 1对DSB修复并不重要，但它与减数分裂特异性MMR异源二聚体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，其调节CO/NCO决定 在后期前期I通过溶解双Holliday连接（dHJ）修复中间体。我们还 鉴定了与FA途径中的另一种解旋酶FANCJ的新型相互作用。当前的目标 建议是阐明SLX 4在I期前期驱动不同DSB修复途径中的作用， 我们假设这种作用取决于它与修复网络中关键参与者的相互作用。在目标1中， 我们将探讨BLM，SLX 4和MUS 81之间的遗传相互作用，特别是问SLX 4是否是 其功能是协调II类事件，或者是否需要促进BLM介导的dHJ溶解。 在目标2中，我们将使用优雅的小鼠模型，在前期I期间鉴定涉及SLX 4的关键功能相互作用。 模型系统地询问SLX 4的每个相互作用伙伴。在目标3中，我们将探讨角色和 FANCJ和SLX 4在前期I期间减数分裂重组中的共依赖性，使用我们的突变小鼠 模型，结合蛋白质组学分析，以了解这两种蛋白质如何在功能上相互作用， 调节CO/NCO决定的背景下，不同的DNA修复途径与它们相互作用。 总的来说，这些研究代表了哺乳动物中SLX 4相互作用组的第一次功能分析。 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。

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.