Single-Molecule FRET TACKLE Reveals Highly Dynamic Mismatched DNA-MutS Complexes

The first step in DNA mismatch repair (MMR) is the recognition of DNA mismatches or nucleotide insertions/deletions (IDLs) by MutS and MutS homologues. To investigate the conformational properties of MutS-mismatch complexes, we used single-molecule fluorescence resonance energy transfer (smFRET) to examine the dynamics of MutS-induced DNA bending at a GT mismatch. The FRET measurements reveal that the MutS GT mismatch recognition complex is highly dynamic, undergoing conformational transitions between many states with different degrees of DNA bending. Due to the complexity of the data, we developed an analysis approach, called FRET TACKLE, in which we combine direct analysis of FRET transitions with examination of kinetic lifetimes to identify all of the conformational states and characterize the kinetics of the binding and conformational equilibria. The data reveal that MutS-GT complexes can reside in six different conformations, which have lifetimes that differ by as much as 20-fold and exhibit rates of interconversion that vary by 2 orders of magnitude. To gain further insight into the dynamic properties of GT-MutS complexes and to bolster the validity of our analysis, we complemented our experimental data with Monte Carlo simulations. Taken together, our results suggest that the dynamics of the MutS mismatch complex could govern the efficiency of repair of different DNA mismatches. Finally, in addition to revealing these important biological implications of MutS DNA interactions, this FRET TACKLE method will enable the analysis of the complex dynamics of other biological systems.