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.
