Elucidation of the Role of Msh4-Msh5 (MutSγ) in Homologous Recombination: A Biophysical and Biochemical Study

In many organisms, MutSγ plays a role in meiotic recombination facilitating crossover formation between homologous chromosomes. Failure to form crossovers leads to improper segregation of chromosomes and aneuploidy, which in humans results in infertility and birth defects. To improve current understanding of MutSγ function, the first part of this thesis investigates the binding affinities and structures of MutSγ in complex with DNA substrates that model homologous recombination intermediates. Our finding demonstrates that Sc Msh4-Msh5 binds Holliday Junction-like substrates, 3' overhangs, single stranded (ss) forks and the D-loop with nanomolar affinity. The weakest binding affinities were detected for an intact duplex and open junction construct. Energy transfer experiments further demonstrate that DNA structure is modulated by the binding interaction with the largest changes associated with substrates containing a ss end. For junction-like intermediates, Msh4-Msh5 binding either stabilizes the existing stacked structure or induces formation of the stacked X conformation. Significantly, we find that upon binding Msh4-Msh5 stacks an open junction construct to the same extent as the standard junction. The different binding modalities observed are suggestive that Msh4-Msh5 not only binds to and stabilizes stacked junctions but also participates in meiotic recombination prior to junction formation by stabilizing single end invasion intermediates. These results suggest that MutSγ stabilizes the stacked X junction conformation which is refractory to branch migration, until resolution by MutLγ.