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

The 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 actinobacterium Corynebacterium glutamicu m, which recognizes mismatched substrates in vitro and creates dsDNA breaks at the mismatch. In this report, a genetic analysis shows that an M. smegmatis Δ nucS strain 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 the M. smegmatis chromosome. 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. glutamicum in vitro, as well as mutation accumulation studies done in M. 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.