Single Molecule Studies of Recombination and Chromosome Pairing in Meiosis

减数分裂中重组和染色体配对的单分子研究

• 批准号: 8400944
• 负责人: Richard Fishel
• 金额: $ 22.88万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-16 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8400944
• 关键词: Binding; Biochemical; Chromosome Pairing; Chromosome Segregation; Chromosomes; Complex; Cruciform DNA; DNA; DNA Double Strand Break; DNA-Binding Proteins; Data; Diploidy; Double Strand Break Repair; Down Syndrome; Evaluation; Event; Filament; Functional disorder; Gene Conversion; Genetic; Genetic Crossing Over; Genetic Recombination; Germ Cells; Goals; Haploidy; Hereditary Disease; Holidays; Homologous Gene; Individual; Infertility; Kinetics; Lead; Link; MLH1 gene; MSH2 gene; MSH4 gene; MSH6 gene; Magnetism; Measures; Mechanics; Mediating; Meiosis; Mismatch Repair; Mutate; Nucleosomes; Optic Chiasm; Process; Resolution; Slide; Spontaneous abortion; System; Testing; Time; base; endonuclease; migration; novel; progenitor; recombinase; reconstitution; repaired; segregation; single molecule

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).

描述（由申请人提供）：减数分裂将正常的细胞二倍体染色体含量降低了一半，以产生单倍体配子。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RAD51重组酶鉴定同源性并在同源染色体之间进行链交换，从而导致D-Loop：Holliday Junction（HJ）交叉的祖细胞。 D-Loop祖细胞HJ中间体通过减数分裂特异性MUTS同源物（MSH）MSH4-MSH5识别，该MSH4-MSH5形成了ATP结合的滑动夹，它包含两个参与的双链DNA链；稳定地连接同源染色体。 MUTL同源物（MLH）MLH1-MLH3特别与MSH4-MSH5相互作用，最终似乎确定了哪些DSB修复事件导致遗传过境。这种看似风险但通常准确的DSB修复进展执行了两项任务：I。）在纺锤体形成和减数分裂I I分离之前和II的同源染色体的稳健配对，以及II。）重新分类是现代遗传学基础的遗传信息。减数分裂染色体配对和隔离的错误是自发性流产以及遗传疾病（例如唐氏综合症（Trisomey 21））的经常原因，RAD51，MSH4-MSH5和MLH1-MLH3之间的合作相互作用尚不清楚。但是，这些相互作用可能是实质性的，因为当去除RAD51时，由RAD51催化的D-Loop中间体不稳定。此外，当MSH4或MSH5突变并且在没有MLH1或MLH3时，染色体配对不正确，染色体分离不正确。导致缺乏可行的配子。该探索性建议的目的是开发新的和定量的探针，以了解RAD51，MSH4-MSH5和MLH1-MLH3之间的复杂相互作用，从而导致减数分裂过程中的染色体配对I。我们已经开发了三种可靠的单分子测量，该测量能够询问和可视化这些基本的MEIISIS I Components Ineper-I Components i Components In实时实时的功能。我们提出了两个具体目的：1。）对重组介导的染色体配对过程中RAD51和MSH4-MSH5之间相互作用的分析，以及2.）分析MSH4-MSH5，MLH1-MLH3和Holliday交界处之间的相互作用。我们似乎是研究这些基本减数分裂染色体成分之间的集合功能的唯一组。我们独特的单分子方法应增强对机械过程的定量理解，从而导致可行的配子形成以及功能障碍之间的细线，从而导致不育和遗传 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，不育和多种遗传疾病（例如唐氏综合症）的原因。关于与减数分裂特异性MUT同源物MSH4-MSH5和MUTL同源物MLH1-MLH3之间的集合功能和MUTL同源物MLH1-MLH3之间的集合功能几乎没有定量生物物理数据，这对于准确的染色体配对和分离至关重要。我们开发了新型的单分子系统，该系统将用于询问这些减数分裂染色体对成分的集合力学，以将清晰的定量值在其功能上放置。