TIFR
Department of Chemical Sciences
School of Natural Sciences

October 23, 2018 at 2.30 pm in AG-69

Title :

Msh4-Msh5 Induced DNA Conformational Changes Provide Insights into its Role in Meiotic Recombination

Abstract :

MutSg 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 MutSg function, we investigated the binding affinities and structures of MutSg in complex with DNA substrates that model homologous recombination intermediates. Our findings demonstrate that Sc Msh4-Msh5 binds Holliday Junction-like substrates, 3' overhangs, single stranded (ss) forks and the D-loop with nanomolar affinity. 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. These results suggest that MutSg stabilizes the stacked X junction conformation, which is refractory to branch migration, possibly until resolution by MutLg. We developed and refined structural models of Msh4-Msh5 interacting with HJ and duplex DNA using homology modelling and molecular dynamics simulations. Importantly, these modelled structures reveal a putative DNA-binding region (DBR), in which the protein makes asymmetric contacts with the junction. We further identified DNA bases and protein residues that are potentially important for binding and recognition. Mutation of these residues or deletion of the DBR results in reduced affinity for HJ and dsDNA. Furthermore, DNA bases predicted to interact with the protein exhibit changes in dynamic motion upon binding that are reduced with mutated protein. Taken together, these results provide significant insight into MutSg binding interactions and the structure-function relationships of these complexes.