Functional Studies of the meiotic Muts Homologs

描述(申请人提供)：大约60%的自发流产似乎是由与受精卵/胎儿相关的染色体异常引起的。这些异常的一个来源是减数分裂过程中染色体不准确的分离。在一些真核生物中的遗传学研究已经明确地将异二聚体特异的MutS同源物MSH4-MSH5与减数分裂I中准确的染色体分离联系在一起。在上一次授予期间，我们在理解这些基因/蛋白质的功能方面取得了重大进展。在这一新的应用中，我们建议继续对人类hMSH4-hMSH5异二聚体蛋白进行生物物理分析。我们将确定与hMSH4-hMSH5功能相关的最相关的异二聚体人类MutL同源物(hMLH1-HPMS 1、hMLH1-hPMS2或hMLH1-hMLH3)。此外，我们将扩大我们对hMSH4-hMSH5减数分裂I功能的生化分析，包括生物相关的重组蛋白(hRAD51；hDMC1；hRAD54；BLM1；HrpA)和联会复合体蛋白(SCP1；SCP2；SCP3)。最后，为了证实我们的体外研究，我们将开发免疫学和肽竞争试剂来检测这些蛋白在减数分裂过程中的细胞定位和相互作用。我们提出了四个具体目标：i)HMSH4-hMSH5的结构域和突变分析确定hMSH4-hMSH5与人MutL异二聚体同系物hMLH1-HPMS1、hMLH1-hPMS2和hMLH1-hMLH3之间的功能相互作用(S)；研究hMSH4-hMSH5与减数分裂特有的染色体配对和重组蛋白之间的功能和生物学相关的相互作用(S)减数分裂特异性MSH/MLH途径组分的鉴定。我们将使用我们所知的这项提议独有的创新方法，例如与表面等离子体共振和总内反射进行比较实时结合，以及通过自由基足迹和质谱分析进行相互作用和表面映射。这些研究将为理解人类MutS和MutL同源物的减数分裂特异功能(S)在准确的染色体分离和维持哺乳动物生育能力方面提供坚实的基础。

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.