Single-Molecule FRET TACKLE Reveals Highly Dynamic Mismatched DNA—MutS Complexest

摘要

The first step in DNA mismatch repair (MMR) is the recognition of DNA mismatches ornucleotide insertions/deletions (IDLs) by MutS and MutS homologues. To investigate the conformationalproperties 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 FRETmeasurements reveal that the MutS–GT mismatch recognition complex is highly dynamic, undergoingconformational transitions between many states with different degrees of DNA bending. Due to thecomplexity of the data, we developed an analysis approach, called FRET TACKLE, in which we combinedirect analysis of FRET transitions with examination of kinetic lifetimes to identify all of the conformationalstates and characterize the kinetics of the binding and conformational equilibria. The data reveal thatMutS–GT complexes can reside in six different conformations, which have lifetimes that differ by as much as20-fold and exhibit rates of interconversion that vary by 2 orders of magnitude. To gain further insight into thedynamic properties of GT –MutS complexes and to bolster the validity of our analysis, we complemented ourexperimental data with Monte Carlo simulations. Taken together, our results suggest that the dynamics of theMutS–mismatch complex could govern the efficiency of repair of different DNA mismatches. Finally, inaddition to revealing these important biological implications of MutS–DNA interactions, this FRETTACKLE method will enable the analysis of the complex dynamics of other biological systems.