MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair.

Background: Nucleotide excision repair and the ATR-mediated DNA damage checkpoint responses are genetically coupled. Results: We have analyzed the basic steps of ATR activation in a biochemically defined system. Conclusion: ATR signaling requires enlargement of the DNA excision gap by EXO1. Significance: The six excision repair factors, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of proteins for ATR-activation upon UV-induced DNA damage.

Background: MutS␣, a heterodimer of MSH2 and MSH6, is essential for DNA mismatch repair. Results: hMsh2 G674A -hMSH6 wt and hMSH2 wt -hMSH6 T1219D mutant proteins fail to efficiently license mismatch-provoked, nick-directed excision. Conclusion: Different defects underlie the apparently similar repair deficiency of these two mutants. Significance: Findings provide new insights into the mechanism of mismatch repair with implications for cancer predisposition and the apoptotic response to DNA damaging agents.

MutS recognizes base–base mismatches and base insertions/deletions (IDLs) in newly replicated DNA. Specific interactions between MutS and these errors trigger a cascade of protein–protein interactions that ultimately lead to their repair. The inability to explain why different DNA errors are repaired with widely varying efficiencies in vivo remains an outstanding example of our limited knowledge of this process. Here, we present single-molecule Förster resonance energy transfer measurements of the DNA bending dynamics induced by Thermus aquaticus MutS and the E41A mutant of MutS, which is known to have error specific deficiencies in signaling repair. We compared three DNA mismatches/IDLs (T-bulge, GT, and CC) with repair efficiencies ranging from high to low. We identify three dominant DNA bending states [slightly bent/unbent (U), intermediately bent (I), and significantly bent (B)] and find that the kinetics of interconverting among states varies widely for different complexes. The increased stability of MutS–mismatch/IDL complexes is associated with stabilization of U and lowering of the B to U transition barrier. Destabilization of U is always accompanied by a destabilization of B, supporting the suggestion that B is a “required” precursor to U. Comparison of MutS and MutS-E41A dynamics on GT and the T-bulge suggests that hydrogen bonding to MutS facilitates the changes in base–base hydrogen bonding that are required to achieve the U state, which has been implicated in repair signaling. Taken together with repair propensities, our data suggest that the bending kinetics of MutS–mismatched DNA complexes may control the entry into functional pathways for downstream signaling of repair.

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.