Eukaryotic DNA Mismatch Repair In Vitro

Mismatch repair corrects biosynthetic errors generated during DNA replication. Mismatch repair deficiency causes a mutator phenotype and directly underlies hereditary nonpolyposis colorectal cancer and some sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae ) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. Here we describe a set of practical protocols for how to prepare the yeast and HeLa cell-free nuclear extracts and site-specific DNA mismatch substrates, and how to carry out the in vitro mismatch repair assay. We validated the yeast cell-free system by the mismatch repair deficient strain (Δmsh2 ) and the complementation assay with purified yeast MutSα.