Research Project
9880871
The process of meiosis is a fundamental cellular process in which the MutS? or Msh4-Msh5 protein is a key component of meiotic recombination in many eukaryotic organisms. MutS? facilitates crossover formation, which is required for the proper segregation of chromosomes and exchange of genetic information. In mammals loss of either subunit leads to infertility. Human aneuploidies are associated with and are a major cause of infertility, miscarriages, congenital birth defects and trisomy disorders such as Down syndrome. Our overall goal in this project is to elucidate structural and functional details of the Saccharomyces cerevisiae (Sc) Msh4-Msh5 protein at a molecular level to obtain a greater understanding of how it functions in the process of meiotic recombination. Our recent work and that of others has pointed to a role for MutS? at an earlier stage of meiotic recombination when single end invasion (SEI) intermediates are formed prior to second end capture and the formation of double Holliday junctions. We are proposing to investigate this critical stage of the crossover pathway by generating structural models of MutS?-SEI intermediates that we will test experimentally using site-directed mutagenesis, energy transfer- based mapping methods and incorporation of fluorescence base analogs to examine changes in single base dynamics as a function of protein binding. In this structure-function analysis, we focus on the role of MutS? in facilitating the progression from SEI intermediates to Holliday junctions and strand exchange. These studies will build on our existing expertise with computational and spectroscopic approaches and our recent isolation and characterization of Sc Msh4-Msh5 expressed in E. coli. We will also explore the coupling of ATPase function with DNA binding through a combination of mutagenesis, equilibrium binding assays, steady state and transient kinetics. In other MutS proteins, mismatch binding stimulates ATP exchange for ADP and triggers conformational changes in the protein and the DNA. We will address whether a similar mechanism is at play in MutS? and if different substrates (such as SEI intermediates versus junction-like intermediates) elicit different responses with respect to ADP release and ATP hydrolysis. Our efforts will be informed by mutation of key residues in the Walker A and B motifs of each subunit, which will reveal if the subunits have differential affinities for ATP or ADP and how that correlates with function. Importantly, our studies will bring molecular level insight to how MutS? functions in meiotic recombination.
