Enhanced Dynamics of Mismatched Base Pairs Associated with Msh2-Msh6 Recognition

Abstract

The DNA mismatch repair (MMR) system guards the integrity of genetic material by scanning and correcting errors in a post-replicative manner. In eukaryotic cells, the initiation of MMR is achieved by recognition by MutS homologs (Msh). The Msh2-Msh6 heterodimer plays this important initial role in recognizing single base mismatches and small insertion/deletion loops (IDL) in MMR. Although Msh2-Msh6 recognizes mismatched DNA with high affinity, the exact mechanism by which Msh2-Msh6 distinguishes different types of mismatched base pairs from a large excess of canonical Watson-Crick base pairs is still unknown. In this study, we use the intrinsic fluorescent probe 6-methylisoxanthopterin (6-MI, guanosine analog) in the context of the ATFAA (F = 6-MI) pentamer sequence where it exhibits enhanced fluorescence, to measure the binding affinity of S. cerevisiae Msh2-Msh6 to different single base pair mismatches. Fluorescence anisotropy measurements reveal the following order for Msh2-Msh6 mismatch bp binding affinity: A:A ≈ A:G > A:C ≈ G:T ≈ +T > T:T ≈ G:G > T:C ≈ G:C. Fluorescence intensity measurements suggest a greater degree of DNA distortion accompanies binding to well-recognized mismatches. We employ Forster resonance energy transfer to measure the bending angle and compare with that observed in MutS/Msh2-Msh6 co-crystal structures. We have also investigated DNA dynamics upon Msh2-Msh6 binding using time-resolved fluorescence spectroscopy. Specific placement of the probe at the mismatch site or adjacent to it reveals significant local motion prior to protein binding. We observe that high affinity binding is associated with those mismatches that exhibit the greatest amount of motion. Protein binding stabilizes mismatch local motion, which is consistent with Phe intercalation at the site, as observed in Msh2-Msh6-DNA co-crystal structures.