Exploring the Role of Msh2‐Msh6 in Processing Holliday Junction DNA

Abstract

Msh2‐Msh6 is a eukaryotic ATP‐dependent mismatch repair (MMR) protein that plays a key role in maintaining the fidelity of the genetic code. The protein recognizes and initiates repair for single base pair mismatches and short insertion deletion loops (IDL). Msh2‐Msh6 functions via a molecular switch mechanism, whereby ATP hydrolysis and ADP/ATP exchange facilitates the binding and release of the protein from the mismatch. Previous research has revealed that the protein binds G:T mismatch DNA with the highest affinity. Interestingly, in‐solution binding assays have revealed that Msh2‐Msh6 binds Holliday Junction DNA, a key recombination intermediate, with 1:1 stoichiometry and a similarly high affinity. However, the functional importance and structural details of this interaction are unclear. Thus far, X‐ray crystallography and Molecular Dynamics (MD) simulations are limited to the protein’s interactions with mismatch DNA only. To address the lack of structural information, we modeled the Msh2‐Msh6‐HJ interaction using the human Msh2‐Msh6 crystal structure in which we have docked in junction DNA instead of the mismatched DNA. The junction DNA used in all experiments and simulations is J3, a non‐migrating junction with 17 base pair long arms. These MD simulations will help refine the structural model and assess significant interactions between the protein and DNA. Furthermore, ATPase assays and equilibrium binding assays are used to elucidate the protein’s ATP hydrolysis activity and binding affinity in the presence of G:T mismatch DNA, J3 junction DNA, and J3 junction DNA containing a G:T mismatch. Preliminary experiments show that relative to the apoprotein’s basal ATPase activity, ATP hydrolysis is weakly stimulated when Msh2‐Msh6 is bound to G:T mismatch and J3. Gel mobility shift assays (GMSA) reveal binding affinities in the range of 9.1±2.6 nM to 23.5±11.8 nM for G:T mismatch, J3 junction, and mismatched J3 junction. These data illustrate that Msh2‐Msh6 not only binds G:T mismatch and J3 with similarly high affinity, but also that introducing a mismatch in J3 does not modulate the protein’s affinity for the junction. In addition, the GMSA data suggest that Msh2‐Msh6‐HJ binding may take on multiple conformations, consistent with previous Förster resonance energy transfer (FRET) calculations that show Msh2‐Msh6 opening the junction after binding.