Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication

Abstract

The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the β-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-β−clamp complex formed via a carboxy-terminal sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system.

New Insights Into DNA Repair Revealed by NucS Endonucleases From Hyperthermophilic Archaea

“…The C. glutamicum EndoMS was further confirmed as the mismatch-specific endonuclease and its function was fully dependent on its physical interaction with the sliding clamp. In vivo, a combination of the C. glutamicum EndoMS gene disruption and a mutation in the dnaE gene can lead to the increased mutation rates, thereby confirming the role of EndoMS in replication error correction (Ishino et al, 2018). In vitro, the C. glutamicum EndoMS can specifically cleave G/T, G/G, and T/T mismatches (Ishino et al, 2018), consistent with the mutation spectrum observed by genome-wide analyses.…”

“…In vivo, a combination of the C. glutamicum EndoMS gene disruption and a mutation in the dnaE gene can lead to the increased mutation rates, thereby confirming the role of EndoMS in replication error correction (Ishino et al, 2018). In vitro, the C. glutamicum EndoMS can specifically cleave G/T, G/G, and T/T mismatches (Ishino et al, 2018), consistent with the mutation spectrum observed by genome-wide analyses. Thus, the bacterial NucS endonucleases resemble their archaeal homologues, and are capable of acting on mismatched bases in DNA to potentially complete the MMR DNA repair pathway.…”

“…Further mutational spectrum data demonstrated that the increased mutation rates are due to elevated levels of base conversion (A:T to G:C conversion or G:C to A:T conversion), which are the mutations created by a typical MMR defect (Castaneda-Garcia et al, 2017). Similar effects were observed in Corynebacterium glutamicum and Streptomyces coelicolor (Ishino et al, 2018;Takemoto et al, 2018). The C. glutamicum EndoMS was further confirmed as the mismatch-specific endonuclease and its function was fully dependent on its physical interaction with the sliding clamp.…”

Specificity and mutagenesis bias of the mycobacterial alternative mismatch repair analyzed by mutation accumulation studies

“…Inactivation of NucS in different actinobacterial species leads to the emergence of hypermutator strains with increased spontaneous mutation rates (4,8,10). Some bacterial isolates with impaired MMR capacity have been detected in nature, suggesting that modulation of MMR activity can be adaptive (11)(12)(13).…”

Bacterial NHEJ: a never ending story

“…Whatever the mechanism, the reduced frequency of the corresponding deletion in a NHEJ‐deficient mutant led to the proposal that DSB generated by MutL Paer are repaired by the NHEJ pathway in this bacterium (Shen et al , ). On the other hand, the majority of archaeal species and many actinobacteria lack MutHLS system but display genes putatively involved in a novel mismatch repair system, called NucS/EndoMS, which has been shown in Corynebacterium glutamicum to cleave both strands at double‐strand DNA mismatched substrate during the repair process (Castañeda‐García et al , ; Ishino et al , ). Whether these induced DSBs are thereafter processed by NHEJ or HR is not yet known, but one can suggest that chromosomes of bacteria possessing a NucS/EndoMS system are more prone to DSBs and consequently to mutations and rearrangements, especially if they also feature a NHEJ machinery, like many actinomycetes.…”