Toward Adding Complexity in Single Molecule FRET Studies of DNA Mismatch Repair

Abstract

Mistakes made during DNA replication are targeted for post-replicative repair by the DNA mismatch repair system. Purified reconstitutions of DNA mismatch repair require multiple protein interactions, and in vivo studies of DNA mismatch repair function have identified layers of spatial and temporal regulation. We have previously developed single molecule FRET experiments for studies of purified in vitro studies of DNA mismatch interactions from Thermus Aquaticus MutS and MutL. In this poster we present our recent progress developing single molecule FRET assays for more complex studies of DNA repair. In particular, we highlight our work using unnatural amino acid engineering and other methods for site-specific labeling of yeast repair proteins, detailed analysis of kinetics to reveal nucleotide regulation of protein conformations, and steps toward studies in live cells.