Joint Molecule Resolution During Meiotic Recombination

项目摘要/摘要 交叉有助于减数分裂过程中准确的染色体分离，这种特殊的细胞分裂 产生配子。我们对超度的理解有一个根本的差距，那就是双霍利迪结点是如何 (Dhj)中间体被具体分解为交叉，以及这一过程是如何被监管的。这 由于在生物化学和细胞学研究dHJ分解方面的挑战，知识差距一直存在 级别。这些障碍在本提案中通过在理解 DHJ拆分机理及生化、遗传和分子试剂的发展 体外和体内研究dHJ拆分的方法。其中包括实现dhj解析 通过一种类似于DNA错配修复的启动步骤的机制，并涉及到对 DHJ由MutLg内切酶激活，由复制钳制的增殖细胞核抗原触发。这些观察结果唤起了一种 定向加载增殖细胞核抗原靶时交叉特定dHJ分辨率的相干模型 MutLγ切割Holliday连接(HJs)两侧的特定DNA链。然后通过以下方式进行解析 HJS在Bloom解旋酶复合体(BLM-TOP3-RMI1/2)驱动的NICK之间的迁移。关键是， 不对称的刻痕图案总是会产生交叉。该项目的长期目标是 了解在减数分裂过程中交叉特异的dhj分解的机制和调节。 通过三个目标。Aim 1将通过以下方式在体外测试交叉特定dHJ解析模型的关键原理 用纯化的人类蛋白和多种含有HJS和HJS的DNA底物重建反应 为增殖细胞核抗原加载站点。在AIM2中，对该模型的预测也将使用无与伦比的 在发芽酵母中可用的分子遗传学工具，并通过新的方法分离dHJ 使用实时遗传学解决步骤和研究相关因素。这个目标将检测MutLγ催化的NICK 在预期的交叉点和准备进行解析的dHJ中；检测未成熟的解析产品 包含单链襟翼和缝隙；确认SGS1-top3-Rmi1(正交系)的交叉解析作用 人类博莱姆复合体)；确定MutLγ对不同的分解途径的影响；以及测试 认为外显子1在交叉位点起作用，以稳定MutLγ催化的NICK。AIM 3将发掘新的见解 通过类似Polo的蛋白激酶cdc5来调节dHJ的分辨率。由磷酸盐确定的候选靶标- 蛋白质组学将在发芽酵母中使用分子遗传学工具进行分析，以了解CDC5是如何触发的 Dhj分辨率。这些目标的结果将提供对机制和监管的前所未有的洞察 在减数分裂期间交叉的可能性。这些发现将与理解与以下相关的病理学密切相关 人类减数分裂，包括不孕不育、流产、先天性疾病和卵巢早衰，以及 预计将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.