Global and Local Conformational Studies of Mismatched Duplex DNA Upon Msh2-Msh6 Binding 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 and thus improves the fidelity of DNA replication by several orders of magnitude. In eukaryotic cells, the initiation of MMR is achieved by recognition of biosynthetic errors by Msh proteins (MutS homologs). Single base mismatches and small insertion/deletion loops (IDL) are recognized by the Msh2-Msh6 heterodimer followed by recruitment of MutL homologs (Mlh/Pms) and this ATP-dependent ternary complex further activates downstream MMR events. The exact mechanism by which Msh2-Msh6 distinguishes a mismatched base pair precisely from a large excess of canonical Watson-Crick base pairs in an efficient manner is still unknown. In this study we explore the specificity of Msh2-Msh6 binding that enables discrimination between different mismatches through measurement of binding affinity and protein-induced bending using fluorescence anisotropy, gel mobility shift assays and Forster Resonance Energy Transfer (FRET). These measurements yield the following order of binding affinity: G:T > +T > G:A > G:C and are in good agreement with previous results. The FRET efficiency of free and Msh2-Msh6-bound mismatched duplex DNA suggests that Msh2-Msh6 bends both +T and G:T duplex DNA relative to G:C homoduplex DNA, which appears to remain relatively straight in the bound form. We have also monitored local DNA base pair dynamics with time-resolved fluorescence intensity and anisotropy spectroscopy measurements. By using a fluorescent nucleoside analog, 6-methylisoxanthopterin incorporated into mismatched DNA, we can explicitly investigate single base pair dynamics. These experiments reveal that protein binding stabilizes the probe placed at the mismatch or located adjacent to the unpaired thymine. This stabilization is relatively local to the mismatch site and is not propagated down the helix.