Functional Studies of the meiotic Muts Homologs

描述（由申请方提供）：大约60%的自然流产似乎是由受精卵/胎儿相关的染色体畸变引起的。这些异常的来源之一是减数分裂减数分裂过程中染色体分离不准确。在许多真核生物中的遗传研究已经清楚地将异二聚体减数分裂特异性MutS同源物MSH 4-MSH 5与减数分裂I中的精确染色体分离联系起来。在上一个授予期，我们在理解这些基因/蛋白质的功能方面取得了重大进展。在本更新申请中，我们建议继续对人hMSH 4-hMSH 5异二聚体蛋白进行生物物理分析。我们将确定与hMSH 4-hMSH 5功能相关的最相关的异源二聚体人类MutL同源物（hMLH 1-hPMS 1、hMLH 1-hPMS 2或hMLH 1-hMLH 3）。此外，我们将扩大我们的hMSH 4-hMSH 5减数分裂I功能的生化分析，包括生物相关的重组蛋白（hRAD 51; hDMC 1; hRAD 54; BLM 1; hRPA）和联会复合体蛋白（SCP 1; SCP 2; SCP 3）。最后，为了证实我们的体外研究，我们将开发免疫和肽竞争试剂来检测这些蛋白质在减数分裂过程中的细胞定位和相互作用。我们提出四个具体目标：（一）hMSH 4-hMSH 5的结构域和突变分析; II.）确定hMSH 4-hMSH 5与异二聚体人MutL同源物hMLH 1-hPMS 1、hMLH 1-hPMS 2和hMLH 1-hMLH 3之间的功能性相互作用; III.）检查hMSH 4-hMSH 5与减数分裂特异性染色体配对和重组蛋白之间的功能和生物学相关相互作用;和IV.）鉴定减数分裂特异性MSH/MLH途径组分。我们将使用创新的方法，据我们所知，这是唯一的建议，如比较实时结合与表面等离子体共振和全内反射，以及相互作用和表面映射通过自由基足迹和质谱分析。这些研究将为理解人类MutS和MutL同源物在精确的染色体分离和维持哺乳动物生育力中的减数分裂特异性功能提供坚实的基础。

DESCRIPTION (provided by applicant): Approximately 60% of spontaneous abortions appear to result from chromosome aberrations associated with the zygote/fetus. One source of these abnormalities is inaccurate chromosome disjunction during the reduction division of meiosis. Genetic studies in a number of eukaryotes has clearly linked the heterodimeric meiosis-specific MutS homologs, MSH4-MSH5, to accurate chromosome segregation in meiosis I. In the last granting period we made significant progress in understanding the function of these genes/proteins. In this renewal application we propose to continue the biophysical analysis of the human hMSH4-hMSH5 heterodimeric proteins. We will determine the most relevant heterodimeric human MutL homolog (hMLH1-hPMS 1, hMLH1-hPMS2, or hMLH1-hMLH3) that is associated with hMSH4-hMSH5 function. In addition, we will expand our biochemical analysis of hMSH4-hMSH5 meiosis I functions to include biologically relevant recombination proteins (hRAD51; hDMC1; hRAD54; BLM1; hRPA) and synaptonemal complex proteins (SCP1; SCP2; SCP3). Finally, to confirm our in vitro studies, we will develop immunological and peptide competition reagent to examine the cellular localization and interaction of these proteins during meiosis. We propose four Specific Aims: I.) domain and mutational analysis of hMSH4-hMSH5; II.) determine the functional interaction (s) between hMSH4-hMSH5 and the heterodimeric human MutL homologs hMLH1-hPMS 1, hMLH1- hPMS2, and hMLH1-hMLH3; III.) examine the functional and biologically relevant interaction(s) between hMSH4-hMSH5 and meiosis-specific chromosome pairing and recombination proteins; and IV.) identification of meiosis-specific MSH/MLH pathway components. We will use innovative methods that to our knowledge are unique to this proposal, such as comparative real-time binding with Surface Plasmon Resonance and Total Internal Reflectance as well as interaction and surface mapping via free-radical footprinting and mass spectral analysis. These studies will provide a substantial foundation for understanding the meiosis-specific function(s) of the human MutS and MutL homologs in accurate chromosome segregation and the maintenance of mammalian fertility.