Single Molecule Studies of Recombination and Chromosome Pairing in Meiosis

描述(申请人提供)：减数分裂使正常细胞的二倍体染色体含量减半，以产生单倍体配子。在这种减数分裂之前，必须配对二倍体染色体同源物。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来修复和重组单个染色体对。DSB修复需要RAD51重组酶来鉴定同源性，并在同源染色体之间执行链交换，从而导致D-环：Holliday Junction(HJ)交叉的祖先。D-环前体HJ中间体被减数分裂特异的MutS同源物(MSH)MSH4-MSH5识别，该中间体形成ATP结合的滑动钳，包含两条参与的双链DNA链；稳定地连接同源染色体。MutL同源(MLH)MLH1-MLH3与MSH4-MSH5特异地相互作用，并最终似乎决定了哪些DSB修复事件导致了遗传交换。这一看似有风险但大致准确的DSB修复进度执行两项任务：i)在纺锤体形成和减数分裂I分离之前，同源染色体的强劲配对，以及II。)基因信息的重新分类，这是现代遗传学的基础。减数分裂染色体配对和分离错误是导致自然流产和唐氏综合征等遗传病的常见原因。RAD51、MSH4-MSH5和MLH1-MLH3之间的协同作用尚不清楚。然而，这些相互作用可能是实质性的，因为当RAD51被移除时，由RAD51催化的D-环中间体是不稳定的。此外，当MSH4或MSH5突变时，染色体配对缺失；当MLH1或MLH3缺失时，染色体分离不能正确发生；导致缺乏可存活的配子。这一探索性提案的目标是开发新的定量探针，以了解RAD51、MSH4-MSH5和MLH1-MLH3之间的复杂相互作用，这些相互作用导致减数分裂I过程中染色体配对。我们已经开发了三种强大的单分子方法，能够实时询问和可视化这些减数分裂I组分的功能。我们提出了两个具体目标：1)分析RAD51与MSH4-MSH5在重组介导的染色体配对过程中的相互作用，以及2.MSH4-MSH5、MLH1-MLH3和Holliday连接的相互作用分析。我们似乎是唯一一个研究这些重要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存活配子形成的机械过程的定量理解，以及导致不孕和遗传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导致大多数流产、不孕和几种遗传病(如唐氏综合症)的原因。关于双链断裂修复成分RAD51与减数分裂特异的MutS同源物MSH4-MSH5和MutL同源物MLH1-MLH3之间的整体功能(S)的定量生物物理数据很少，这对于精确的染色体配对和分离是必不可少的。我们已经开发了新的单分子系统，将被用来询问这些减数分裂染色体配对组件的整体机制，以对它们的功能进行明确的量化(S)。

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).