Single Molecule Studies of Recombination and Chromosome Pairing in Meiosis

描述（由申请方提供）：减数分裂将正常细胞二倍体染色体含量减少一半，以产生单倍体配子。二倍体同源染色体必须在减数分裂前配对。配对涉及在整个染色体中引入数百个DNA双链断裂（DSB），然后使用同源染色体同源物来修复和重新组装单个染色体对。DSB修复需要RAD 51重组酶来鉴定同源性并在同源染色体之间进行链交换，从而导致D环：霍利迪连接（HJ）交换的祖先。D-环祖细胞HJ中间体被减数分裂特异性MutS同源物（MSH）MSH 4-MSH 5识别，其形成ATP结合的滑动夹，其包含参与的双链DNA链;稳定地连接同源染色体。MutL同源物（MLH）MLH 1-MLH 3与MSH 4-MSH 5特异性相互作用，并最终似乎决定哪一个DSB修复事件导致遗传交换。这种看似有风险但通常准确的DSB修复进展执行两个任务：I.在纺锤体形成和减数分裂I分离之前同源染色体的稳健配对，和II.）遗传信息的重组是现代遗传学的基础。减数分裂染色体配对和分离中的错误是自发流产以及遗传疾病如唐氏综合征（21三体）的常见原因，RAD 51、MSH 4-MSH 5和MLH 1-MLH 3之间的协同相互作用是未知的。然而，这些相互作用可能是实质性的，因为当去除RAD 51时，由RAD 51催化的D-环中间体是不稳定的。此外，当MSH 4或MSH 5突变时，染色体配对不存在，当MLH 1或MLH 3不存在时，染色体分离不正确;导致缺乏可存活的配子。这项探索性建议的目标是开发新的定量探针，以了解在减数分裂I期间导致染色体配对的RAD 51，MSH 4-MSH 5和MLH 1-MLH 3之间的复杂相互作用。我们已经开发了三个强大的单分子措施，能够询问和可视化这些基本的减数分裂I组件的功能，在实时。我们提出了两个具体目标：1。在重组介导的染色体配对过程中分析RAD 51和MSH 4-MSH 5之间的相互作用，和2.）MSH 4-MSH 5、MLH 1-MLH 3和Holliday连接之间的相互作用的分析。我们似乎是唯一一个研究这些基本的减数分裂染色体配对组件之间的整体功能的小组。我们独特的单分子方法应该加强对导致可行配子形成的机械过程的定量理解，以及导致不育和遗传性疾病的功能障碍之间的细微差别。 疾病 公共卫生相关性：减数分裂染色体分离的错误是大多数流产、不育和几种遗传疾病如唐氏综合症的原因。关于双链断裂修复组分RAD 51与减数分裂特异性MutS同源物MSH 4-MSH 5和MutL同源物MLH 1-MLH 3之间的整体功能的定量生物物理数据非常少，这对于精确的染色体配对和分离是必需的。我们已经开发了新的单分子系统，将用于询问这些减数分裂染色体配对组件的整体力学，以明确其功能的定量值。

DESCRIPTION (provided by applicant): Meiosis reduces the normal cellular diploid chromosome content by half to create haploid gametes. Diploid chromosome homologs must be paired prior to this reduction division. Pairing involves the introduction of several hundred DNA double stranded breaks (DSBs) throughout the chromosomes, which then use the cognate chromosome homolog to repair and reassemble individual chromosome pairs. DSB repair requires the RAD51 recombinase to identify homology and perform strand exchange between homologous chromosomes that results in a D-loop: the progenitor to a Holliday Junction (HJ) crossover. D-loop progenitor HJ intermediates are recognized by the meiosis-specific MutS homologs (MSH) MSH4-MSH5, which form ATP-bound sliding clamps that embraces both the participating duplex DNA strands; stably linking the homologous chromosomes. The MutL homologs (MLH) MLH1-MLH3 specifically interact with MSH4-MSH5 and ultimately appear to determine which of the DSB repair events results in genetic crossing-over. This seemingly risky but generally accurate DSB repair progression performs two tasks: I.) the robust pairing of homologous chromosomes prior to spindle formation and meiosis I segregation, and II.) The reassortment of genetic information that is the basis of modern genetics. Mistakes in meiosis chromosome pairing and segregation are the frequent cause of spontaneous miscarriages as well as genetic diseases such as Down syndrome (Trisomey 21) the cooperative interactions between RAD51, MSH4-MSH5 and MLH1-MLH3 is unknown. However, these interactions are likely to be substantial since the D-loop intermediates catalyzed by RAD51 are unstable when RAD51 is removed. Moreover, chromosome pairing is absent when MSH4 or MSH5 are mutated and chromosome segregation does not occur properly when MLH1 or MLH3 are absent; leading to a lack of viable gametes. The goal of this exploratory proposal is to develop new and quantitative probes to understand the complex interactions between RAD51, MSH4-MSH5 and MLH1-MLH3 that result in chromosome pairing during meiosis I. We have developed three robust single molecule measures capable of interrogating and visualizing the functions of these essential meiosis I components in real-time. We propose two specific aims: 1.) analysis of the interaction between RAD51 and MSH4-MSH5 during recombination mediated chromosome pairing, and 2.) analysis of the interactions between MSH4-MSH5, MLH1-MLH3 and Holliday Junctions. We appear to be the only group examining the ensemble functions between these essential meiosis chromosome-pairing components. Our unique single molecule approach should enhance the quantitative understanding of mechanical processes that lead to viable gamete formation as well as the fine line between dysfunctions that lead to infertility and genetic disease. PUBLIC HEALTH RELEVANCE: Errors in meiosis chromosome segregation are responsible for a majority of miscarriages, infertility and several genetic diseases such as Down syndrome. There is very little quantitative biophysical data regarding the ensemble function(s) between the double strand break repair component RAD51 with the meiosis specific MutS homologs MSH4-MSH5 and MutL homologs MLH1-MLH3, that are essential for accurate chromosome pairing and segregation. We have developed novel single molecule systems that will be used to interrogate the ensemble mechanics of these meiosis chromosome-pairing components to place clear quantitative values on their function(s).