Joint Molecule Resolution During Meiotic Recombination

项目总结/摘要 交换促进了减数分裂过程中染色体的精确分离，减数分裂是一种特殊的细胞分裂， 产生配子。在我们对交叉的理解中，一个根本的差距是双霍利迪结 (dHJ)中间体被具体分解为交叉，以及如何调节这一过程。这 由于在生物化学和细胞生物学领域研究dHJ分辨率的挑战， 程度.本提案通过在理解 dHJ分解的机制以及生化、遗传和分子试剂的开发， 研究dHJ在体外和体内的分辨率的方法。其中包括实现dHJ分辨率 通过一种类似于DNA错配修复起始步骤的机制， dHJ通过MutLg核酸内切酶，通过复制钳PCNA进行标记。这些观察唤起了一个 交叉特异性dHJ分辨率的相干模型，其中PCNA靶向的方向特异性加载 MutLγ切割Holliday连接（HJ）两侧的特异性DNA链。然后通过以下方式解决问题 由Bloom解旋酶复合物（BLM-TOP 3-RMI 1/2）驱动的HJ穿过切口的迁移。重要的是， 刻痕的不对称图案总是产生交叉。该项目的长期目标是 了解减数分裂过程中交叉特异性dHJ分解的机制和调节，将继续进行 通过三个目标。目的1将通过以下方式在体外测试交叉特异性dHJ分辨率模型的关键原则： 使用纯化的人蛋白质和多种含有HJ的DNA底物重建反应， PCNA的加载位点。在Aim 2中，模型的预测也将使用无与伦比的 芽殖酵母中可用的分子遗传学工具，通过分离dHJ的新方法得到增强 解析步骤，并使用实时遗传学研究相关因素。该目的将检测MutLγ催化的缺口 在预期交叉研究中心和准备消退的dHJ中;检测不成熟的消退产物 包含单链活瓣和缺口;确认Sgs 1-Top3-Rmi 1（直系同源物）的交叉分辨作用 人BLM复合物）;确定MutLγ对替代拆分途径的影响;并测试 认为Exo 1在交叉位点起稳定MutLγ催化的切口的作用。Aim 3将利用新的见解 通过polo样激酶Cdc 5调节dHJ分解。通过磷酸- 蛋白质组学将在芽殖酵母中使用分子遗传学工具进行分析，以了解Cdc 5如何触发 dHJ分辨率。这些目标的结果将为机制和监管提供前所未有的见解 在减数分裂过程中交换。这些发现将有助于理解与糖尿病相关的病理学。 人类减数分裂，包括不育、流产、先天性疾病和卵巢功能不全，以及 有望定义与染色体生物学广泛相关的范例。

PROJECT SUMMARY/ABSTRACT Crossing over facilitates accurate chromosome segregation during meiosis, the specialized cell division that produces gametes. A fundamental gap in our understanding of crossing over is how double-Holliday junction (dHJ) intermediates are specifically resolved into crossovers and how this process is regulated. This knowledge gap has persisted because of challenges to study dHJ resolution at the biochemical and cellular levels. These impediments are overcome in this proposal through breakthroughs in understanding the mechanism of dHJ resolution and the development of biochemical, genetic, and molecular reagents and approaches to study dHJ resolution in vitro and in vivo. These include the realization that dHJ resolution occurs through a mechanism that resembles the initiation steps of DNA mismatch repair and involves the nicking of dHJs by the MutLg endonuclease, trigged by the replicative clamp PCNA. These observations evoke a coherent model for crossover-specific dHJ resolution in which orientation-specific loading of PCNA targets MutLγ to incise specific DNA strands on both sides of the Holliday junctions (HJs). Resolution then occurs via migration of the HJs across the nicks driven by the Bloom helicase complex (BLM-TOP3-RMI1/2). Critically, the asymmetric pattern of nicks always yields a crossover. The long-term objective of this project, to understand the mechanism and regulation of crossover-specific dHJ resolution during meiosis, will be pursued through three aims. Aim 1 will test the key tenets of the crossover-specific dHJ resolution model in vitro by reconstituting the reaction using purified human proteins and a variety of DNA substrates containing HJs and loading sites for PCNA. In Aim2, predictions of the model will also be tested in vivo using the unrivaled suite of molecular-genetics tools available in budding yeast, augmented by new approaches to isolate the dHJ resolution step and study pertinent factors using real-time genetics. This aim will detect MutLγ-catalyzed nicks at prospective crossover sites and in dHJs poised for resolution; detect immature resolution products containing single-stranded flaps and gaps; confirm the crossover resolution role of Sgs1-Top3-Rmi1 (ortholog of the human BLM complex); determine the influence of MutLγ on alternative resolution pathways; and test the idea that Exo1 functions at crossover sites to stabilize MutLγ-catalyzed nicks. Aim 3 will exploit new insights into the regulation of dHJ resolution by polo-like kinase, Cdc5. Candidate targets identified by phospho- proteomics will be analyzed using molecular-genetics tools in budding yeast to understand how Cdc5 triggers dHJ resolution. The results of these aims will provide unprecedented insight into the mechanism and regulation of crossing over during meiosis. These findings will be germane to understanding pathologies associated with human meiosis, including infertility, miscarriage, congenital disease, and premature ovarian insufficiency, and are expected to define paradigms that are broadly relevant for chromosome biology.