Joint Molecule Formation During Recombination

描述(由申请人提供)： 摘要同源重组是一种依赖于模板的DNA修复过程，支持DNA复制和基因组稳定性，对减数分裂过程中的染色体分离是必不可少的。基本的重组反应是通过同源配对和DNA链交换在断裂的染色体末端和同源模板之间形成联合分子(JM)中间体。JMS以不同的方式成熟和解决，以生产具有或不具有关联交叉的产品。这项建议将调查在减数分裂过程中调节JMS形成和分解的因素在体内的作用。一般的假设是，专门的JM加工因素受到不同的空间和时间调节。将体内功能分配给特定的JM加工因子并了解它们在重组过程中如何相互作用仍然是具有挑战性的问题，重叠/补偿活动以及监测分子表型的专门分析的需要使这些问题变得复杂。监测体内重组的化学步骤的DNA检测将构成这项研究的基石。有三个不同的目标：AIM1。探讨翻译后修饰对促交叉因子的调节作用。我们已经确定了三个减数分裂特异因子上的磷酸化位点，它们稳定了JMS并促进了交叉。这些因子是DNA解旋酶Mer3，与DNA错配修复因子相关的MutS？复合体，以及相扑E3连接酶Zip3。在这方面，我们的目标是了解这些磷酸化的功能后果，并确定负责的激酶。AIM2.确定促进JMS交叉特定分辨率的因素。我们已经确定了促进将JMS偏颇地分解为交叉的因素。在这一目标中，我们将进一步确定这些因素的特征，以便：(1)确定交接所需的活动和相互作用；(2)证明具体因素在联合专家委员会决议时起作用；(3)了解如何 Polo-Kinase，CDC5，激活交叉特异的分辨率。AIM3.了解Smc5/6复合体在减数分裂过程中如何调节JMS的加工。我们已经证明，SMC家族复合体Smc5/6既调节JMS的形成，也调节JMS的分解。我们将解决这样的想法，即smc5/6突变体形成不能被分解酶处理的病理性JM结构。我们还将探讨Smc5/6复合体在JM形成和JM解析中的时间不同作用是通过调节不同的伙伴蛋白来调节的，重点是DNA解旋酶Mph1和SGs1。这些研究的结果将对理解所有生物体中的减数分裂重组具有广泛的相关性，对于识别使交叉和同源分离过程变得极其准确的减数分裂过程尤其重要。

DESCRIPTION (provided by applicant): SUMMARY Homologous recombination is a template-dependent DNA repair process that underpins DNA replication and genome stability, and is essential for chromosome segregation during meiosis. The fundamental recombination reaction is the formation of Joint Molecule (JM) intermediates via homologous pairing and DNA strand-exchange between a broken chromosome end and a homologous template. JMs are matured and resolved in distinct ways to produce products with or without an associated crossover. This proposal will investigate the in vivo roles of factors that regulate the formation and resolution of JMs during meiosis. The general hypothesis is that specialized JM processing factors are subject to distinct spatial and temporal regulation. Assigning in vivo functions to specific JM processing factors and understanding how they interact during recombination remain challenging issues, which are complicated by overlapping/compensatory activities and the need for specialized assays to monitor molecular phenotypes. DNA assays to monitor the chemical steps of recombination in vivo will form the cornerstone of this investigation. There are three distinct aims: AIM1. To investigate the regulation of pro-crossover factors by post-translational modification. We have identified phosphorylation sites on three meiosis-specific factors that stabilize JMs and promote crossing-over. These factors are the DNA helicase, Mer3, the MutS¿ complex related to DNA mismatch repair factors, and a SUMO E3 ligase, Zip3. In this, aim our goal is to understand the functional consequences of these phosphorylations and identify the responsible kinases. AIM2. To characterize factors that promote crossover-specific resolution of JMs. We have identified factors that promote biased resolution of JMs into crossovers. In this aim, we will further characterize these factors in order to: (i) identify activities and interactions required for crossng-over; (ii) demonstrate that specific factors act at the time of JM resolution; (iii) understand how polo-kinase, Cdc5, activates crossover-specific resolution. AIM3. To understand how the Smc5/6 complex regulates processing of JMs during meiosis. We have shown that the SMC-family complex, Smc5/6 regulates both the formation and resolution of JMs. We will address the idea that smc5/6 mutants form pathological JM structures that cannot be processed by resolving enzymes. We will also investigate the general theme that the temporally distinct roles of the Smc5/6 complex in JM formation and JM resolution are mediated through regulation of distinct partner proteins, with a focus on the DNA helicases Mph1 and Sgs1. Results obtained from these studies will be broadly relevant for understanding meiotic recombination in all organisms and will be especially important for discerning meiotic processes that make crossing-over and homolog disjunction incredibly accurate processes.