| Mismatch repair(MMR)is a critical DNA repair pathway that ensures DNA replication fidelity,mutation avoidance,and genome stability.MMR in bacteria and eukaryotes has been thoroughly studied at both the molecular and mechanistic levels.MutS,MutL and their homologs are the central proteins involved in bacterial and eukaryotic MMR,however,no MutS and MutL homologs has been reported in majority species of Archaea.Intriguingly,the quest for an MMR system in these organisms led to the discovery of a novel Mismatch Specific Endonuclease(EndoMS)in archaea(Euryarchaeota)and some actinobacteria,a novel EndoMS-mediated non-canonical MMR pathway.In the current study,we report the in vitro mismatch DNA cleavage activity and in vivo functional analysis of the homolog of EndoMS(SisEndoMS)from Sulfolobus islandicus REY15A,a genetically tractable crenarchaeon.The biochemical mismatch DNA cleavage assay revealed that SisEndoMS is active against dsDNA having G/T,G/G,and T/T mismatches,but not against C/T,A/G,C/C,A/C,and A/G mismatches.Interestingly,SisEndoMS was also found active against dsDNA having A/G and C/T mismatches,when Mn2+instead of Mg2+was used as the co-factor in the reaction system.The mismatch DNA cleavage activity of SisEndoMS was also stimulated by the heterotrimeric proliferating cell nuclear antigen(PCNA).The cleavage activity enhancement by Mn2+and PCNA indicates that EndoMS activity can be regulated by ion cofactors and accessory proteins.Like TkoEndoMS,SisEndoMS cleaves the third phosphodiester bond on the 5' side of the mismatched base on both strands,resulting in the formation of a cohesive end with a 5-nucleotide long 5' protrusion.SisEndoMS can also cleave uracil(U)-containing double-stranded DNA,implying that it is involved in the repair of uracil-containing dsD-NA caused by deamination,a common DNA damage in living organisms.In the genetic study,the spontaneous mutation rate of the wild type strain REY15A and endoMS knockout mutant(?endoMS)resistant to 5-fluoroorotic acid(5-FOA)was compared.Our finding demonstrated that ?endoMS had a much higher spontaneous rate of mutation(5.06×10-3)as compared to the wild type(4.6×10-6).The mutation accumulation study revealed that the knockout mutant had a higher mutation frequency than the wild type and in comparison,to transversion mutations,transition mutations were found to be dominant.The mutation accumulation analysis suggested that SisEndoMS is responsible for the avoidance of mutation in the hyperthermophile.To evaluate the underlying mechanism of SisEndoMS,we examined the growth,cell morphology,DNA content,and transcriptome of the overexpression strains of EndoMS(wild type),catalytic deficient and/or catalytic and DNA binding deficient mutants of EndoMS.Overexpression of SisEndoMS(wild type)in S.islandicus caused growth retardation and irregular cell morphology,similar to the strains overexpressing Holliday junction endonucleases,Hje or Hjc.According to the transcriptomic study.SisEndoMS overexpression resulted in upregulation of distinct genes,including the CRISPR-Cas ?B mechanism,methyltransferases,and glycosyltransferases,all of which are primarily localized to a single chromosome segment.Our findings suggest that EndoMS proteins are involved in a noncanonical DNA repair pathway in archaea.The mechanism of this EndoMS-mediated non-canonical mismatch repair pathway is discussed... |
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