Investigating the Role of Msh4-Msh5 ATPASE Activity During Homologous Recombination

Abstract

Msh4-Msh5 or MutSgamma is a protein belonging to the MutS family of DNA repair proteins that are mainly involved in post-replicative mismatch repair for the purpose of maintaining genome integrity. Msh4-Msh5 has a different role in which the protein facilitates crossover formation during meiotic recombination in many eukaryotic organisms. Failure to form crossovers results in improper segregation of chromosomes during meiosis, which can lead to infertility and birth defects. Understanding the structural and functional intricacies of this protein will help to elucidate how it functions in crossover formation and meiotic recombination. Previously, our lab has shown that Msh4-Msh5 binds to Holliday junctions (HJ) and HJ-like structures such as 3’ overhangs, single-stranded forks, and D-loops with high affinity, as opposed to DNA duplexes to which the protein binds with much weaker affinity. Generally, our FRET measurements show Msh4-Msh5 displaces the single strand in single-strand containing substrates and induces stacked junction-like structures for substrates more closely resembling Holliday junctions. We are investigating how the presence of nucleotides affects the DNA binding properties of S. cerevisiae Msh4-Msh5. Our approach uses a combination of fluorescently-labeled substrates and nucleotides to determine dissociation constants in the presence of different recombination intermediates while also examining protein-induced conformational changes. We are pairing these results with kinetic studies of ATP hydrolysis in the presence of the same DNA recombination intermediates to gain a clearer understanding of how Msh4-Msh5 ATPase activity is coupled with DNA and protein conformational changes. Through this work, we are investigating the potential molecular switch mechanism of Msh4-Msh5 by which other members of the MutS family have been show to function, while comparing our results from studying S. cerevisiae Msh4-Msh5 to previous results reporting on human Msh4-Msh5.