Single Molecule Fluorescence and Atomic Force Microscopy Studies of DNA Repair

DNA polymerases that are responsible for replication make approximately one error for every 107 bases copied, but the human genome contains ∼6 billion bases, which results in ∼600 errors per round of replication. The DNA mismatch repair (MMR) system corrects these DNA synthesis errors that occur during replication. MMR is initiated by the highly conserved MutS and MutL homologs, which are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. MutS homologs initiate repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutL homologs. This complex signals the initiation of excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. We have been using a combination of atomic force microscopy (AFM) and single molecule fluorescence to characterize the stoichiometries and the conformational and dynamic properties of MutS and MutL homologs and their assembly on DNA containing a mismatch. We have also developed a new dual resonance frequency enhanced electrostatic force microscopy (DREEM), in which we simultaneously collect the AFM topographic image and an image of the electrostatic potential of the surface. The DREEM images reveal the path of DNA inside individual protein-DNA complexes, yielding unprecedented details about DNA conformations within simple and complicated complexes. I will discuss our studies on the assembly of MutS and MutL homologs on mismatches, with a focus on how AFM, DREEM, and single-molecule fluorescence can be powerful tools to study the stoichiometries, conformations, and dynamic assembly of multi-component complexes.