Complex formation in DNA mismatch repair

The conserved DNA mismatch repair machinery corrects errors of the DNA replication machinery or responds to several kinds of DNA damage to initiate repair or signal to other pathways. An early event in DNA mismatch repair (MMR) is the mismatch-provoked formation of a MutS-MutL-DNA complex. This complex is believed to coordinate the subsequent excision repair reaction. In Escherichia coli this requires the sequential activation of the MutH endonuclease, UvrD helicase and one of several exonucleases. Despite decades of work little is known about the structure and dynamics of the protein complexes formed during this process. To analyze the individual steps in MMR, we established a system using Förster Resonance Energy Transfer (FRET) which allows monitoring the formation of protein-DNA and protein-protein complexes in real time in solution. Several DNA, MutS, MutL and MutH variants were generated and site-specifically labeled with various fluorophores. Binary complex formations were monitored using both equilibrium and pre-steady-state fluorescence assays (e.g. stopped-flow technique). We are now in a position to follow individual steps of the DNA mismatch repair machinery using time resolved techniques with nanometer precision. Currently, kinetic parameters of individual steps are determined to develop a kinetic model of the mismatch repair process and to analyze the molecular mechanisms of MMR down to the single-molecule level.