BPS2026 – Investigation of the MutSα interaction with recombination intermediates: An alternate binding mechanism?

DNA mismatch repair (MMR) is a universally conserved pathway essential for maintaining genome integrity, primarily through correction of single base mismatches and insertion/deletion loops generated during DNA replication. In eukaryotes, the MutS homolog, MutSα, initiates this process by recognizing post-replication mismatches via a conserved Phe-X-Glu motif. Additionally, loss of key MMR proteins can lead to increased recombination between divergent sequences, suggesting a role in regulation. Our findings reveal that Sc Msh2-Msh6 binds to Holliday junctions with an affinity comparable to that observed for heteroduplex DNA. However, binding to HJs does not appear to depend on the canonical Phe-X-Glu motif, suggesting an alternative recognition mechanism or binding interface. To explore its role in recombination, we tested the binding interactions between Sc Msh2-Msh6 and the displacement loop (D-loop) and D-loop containing an invading strand. Preliminary results suggest that MutSα binding affinity is influenced by the presence of an invading strand, where Sc Msh2-Msh6 demonstrated higher affinity for D-loops containing an invading strand relative to the D-loop alone. Mass spectrometric analyses of MutSα with and without HJ DNA points to substrate-dependent conformational changes and suggest an alternate binding site for recombination intermediates. Comparative analysis of free versus DNA-bound Sc Msh2-Msh6 revealed the presence of peptides with altered mass profiles upon DNA binding. These and other results will be presented to support our findings of a secondary DNA binding site on MutS that is specifically for recombination intermediates.