Recognition of recombination intermediates by the mismatch repair protein Msh2-Msh6.

Abstract

The mismatch repair (MMR) protein MutSα (Msh2-Msh6), known for improving the fidelity of DNA replication, also improves the fidelity of homologous recombination. Msh2-Msh6 and its associated proteins are responsible for suppressing homologous recombination when excessive base mispairing is present between the participating chromosomes. In MMR, Msh2-Msh6 recognizes small insertion/deletion loops and single base pair mismatches while scanning DNA post replication. Interestingly, Sc Msh2-Msh6 binds to Holliday Junctions (HJs) with an affinity comparable to that of mismatched duplex DNA (Kd =15 nM). We study the Msh2-Msh6-HJ binding interaction to gain insight into the mechanistic role of Msh2-Msh6 during the process of homologous recombination. In our previous study we examined the dynamics of single base mismatches and the changes associated with Msh2-Msh6 binding using the fluorescent DNA base analog 6-MI and time-resolved fluorescence methods. Here, we employ a similar strategy to study the dynamics of single bases within HJs alone and upon interaction with Msh2-Msh6. 6-MI probes incorporated into the center and arms of the junction only exhibit perturbed dynamics in the center location upon Msh2-Msh6 binding, suggesting protein binding takes place at the junction center. Successful UV-photo crosslinking of Msh2-Msh6 to HJs containing 5-bromouracil at the junction center further supports this finding. Base dynamics at the center of the junction resemble those of the mismatch, which possibly drives Msh2-Msh6 recognition. Interestingly, introduction of a mismatch in the arms of the HJ shifts the Msh2-Msh6 binding from the center of HJ to the mismatch location despite the similar binding affinities. Additionally, a molecular model of the Msh2-Msh6-HJ interaction has been created and subjected to molecular dynamics simulations to gain further structural insights with particular focus on how the DNA binding domain interacts with HJs compared to mismatched duplex DNA.