Mycobacterium smegmatisNucS-promoted DNA mismatch repair involves limited resection by a 5’-3’ exonuclease and is independent of homologous recombination and NHEJ

Abstract

AbstractThe MutSL mismatch repair (MMR) system in bacteria and eukaryotes correct mismatches made at the replication fork to maintain genome stability. A novel MMR system is represented by the EndoMS/NucS endonuclease from actinobacteriumCorynebacterium glutamicum, which recognizes mismatched substrates in vitro and creates dsDNA breaks at the mismatch. In this report, a genetic analysis shows that anM. smegmatisΔnucSstrain could be complemented by expression of wild type NucS protein, but not by one deleted of its last five amino acids, a region predicted to be critical for binding to the β-clamp at the replication fork. Oligo-recombineering was then leveraged to deliver defined mismatches to a defective hygromycin resistance gene on theM. smegmatischromosome. We find that NucS recognizes and repairs G-G, G-T, and T-T mismatches in vivo, consistent with the recognition of these same mismatches in C.glutamicumin vitro, as well as mutation accumulation studies done inM. smegmatis. Finally, an assay that employs an oligo that promotes the generation of two mismatches in close proximity on the chromosome shows that a NucS-promoted cut is processed by a 5’–3’ exonuclease and that NucS-promoted MMR is independent of both homologous recombination and non-homologous end-joining.