Endonuclease-independent DNA mismatch repair processes on the lagging strand

DNA mismatch repair (MMR) pathways coordinate the excision and re-synthesis of newly-replicated DNA if a mismatched base-pair has been identified by protein MutS or MutS homologues (MSHs) after replication. DNA excision during MMR is initiated at single-strand breaks (SSBs) in vitro, and several redundant processes have been observed in reconstituted systems which either require a pre-formed SSB in the DNA or require a mismatch-activated nicking endonuclease to introduce a SSB in order to initiate MMR. However, the conditions under which each of these processes may actually occur in living cells have remained obscured by the limitations of current MMR assays. Here we use a novel assay involving chemically-modified oligonucleotide probes to insert targeted DNA ‘mismatches’ directly into the genome of living bacteria to interrogate their replication-coupled repair processes quantitatively in a strand-, orientation-, and mismatched nucleotide-specific manner. This 'semi-protected oligonucleotide recombination’ (SPORE) assay reveals direct evidence in Escherichia coli of an efficient endonuclease-independent MMR process on the lagging strand—a mechanism that has long-since been considered for lagging-strand repair but never directly shown until now. We find endonuclease-independent MMR is coordinated asymmetrically with respect to the replicating DNA—directed primarily from 3′- of the mismatch—and that repair coordinated from 3′- of the mismatch is in fact the primary mechanism of lagging-strand MMR. While further work is required to explore and identify the molecular requirements for this alternative endonuclease-independent MMR pathway, these findings made possible using the SPORE assay are the first direct report of this long-suspected mechanism in vivo.