Single Molecule Studies of Recombination and Chromosome Pairing in Meiosis

描述（由申请人提供）：减数分裂将正常细胞二倍体染色体的含量减少一半，产生单倍体配子。二倍体染色体同源物必须在此还原分裂之前配对。配对涉及在染色体中引入数百个DNA双链断裂（DSBs），然后使用同源染色体同源物修复和重组单个染色体对。DSB修复需要RAD51重组酶识别同源性并在同源染色体之间进行链交换，从而产生d环：Holliday结（HJ）交叉的前身。d环前体HJ中间体被减数分裂特异性MutS同源物（MSH） MSH4-MSH5识别，它们形成atp结合的滑动夹，包含参与的双链DNA链；稳定地连接同源染色体。MutL同源物（MLH） MLH1-MLH3特异性地与MSH4-MSH5相互作用，并最终决定哪一个DSB修复事件导致遗传交叉。这种看似危险但通常准确的DSB修复过程执行两个任务：1)在纺锤体形成和减数分裂之前同源染色体的牢固配对；遗传信息的重新组合是现代遗传学的基础。减数分裂染色体配对和分离错误是自然流产以及唐氏综合症（21三体）等遗传疾病的常见原因，RAD51、MSH4-MSH5和MLH1-MLH3之间的合作相互作用尚不清楚。然而，这些相互作用可能是实质性的，因为当RAD51被去除时，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突变时染色体分离不正常；导致缺乏可存活的配子。本探索性建议的目标是开发新的定量探针，以了解RAD51， MSH4-MSH5和MLH1-MLH3之间导致减数分裂I期间染色体配对的复杂相互作用。我们已经开发了三种强大的单分子测量方法，能够实时查询和可视化这些重要减数分裂I组分的功能。我们提出了两个具体目标：1)分析重组介导的染色体配对过程中RAD51与MSH4-MSH5之间的相互作用；2)分析MSH4-MSH5、MLH1-MLH3和Holliday连接之间的相互作用。我们似乎是唯一一组研究这些基本减数分裂染色体配对组件之间的集合功能。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的功能障碍之间的细线

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).