Global and Local Conformation of Mismatched Duplex DNA Upon MSH2-MSH6 Binding Studied by Steady-State and Time-Resolved Fluorescence

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 of biosynthetic errors by Msh proteins (MutS homologs). The recognition of single base mismatches and small insertion/deletion loops (IDL) by the Msh2-Msh6 heterodimer is an important initial event in the MMR pathway. Several studies have suggested that the prokaryotic MutS homodimer and its eukaryotic counterpart Msh2-Msh6 bind duplex DNA containing a mismatch or IDL with a higher affinity than homoduplex DNA. However, 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 are utilizing DNA duplexes containing 6-methylisoxanthopterin (6-MI) a fluorescent nucleoside analog to measure the binding affinity of S. cerevisiae Msh2-Msh6 to different single base pair mismatches. For greater sensitivity this analog is incorporated into a pentamer sequence ATFAA (F = 6-MI), which exhibits enhanced fluorescence. Fluorescence anisotropy measurements performed with these intrinsically labeled duplexes reveal the following order for Msh2-Msh6 binding affinity to DNA mismatches: G:T > +T ≈ G:A > G:C. This order is consistent with previously reported affinities measured in the gel. We have further 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 those mismatches with the highest affinity exhibit the greatest amount of motion. Protein binding stabilizes mismatch local motion, which is potentially consistent with Phe intercalation at the site, as observed in Msh2-Msh6-DNA co-crystal structures.