ABSTRACT
In eukaryotes and most bacteria, the MutS1/MutL-dependent mismatch repair system (MMR) corrects DNA mismatches that arise as replication errors. MutS1 recognizes mismatched DNA and stimulates the nicking endonuclease activity of MutL to incise mismatch-containing DNA. In archaea, there has been no experimental evidence to support the existence of the MutS1/MutL-dependent MMR. Instead, it was revealed that a large part of archaea possess mismatch-specific endonuclease EndoMS, indicating that the EndoMS-dependent MMR is widely adopted in archaea. However, some archaeal genomes encode MutS1 and MutL homologs, and their molecular functions have not been revealed. In this study, we purified and characterized recombinant MutS1 and the C-terminal endonuclease domain of MutL from a methanogenic archaeon Methanosaeta thermophila (mtMutS1 and the mtMutL CTD, respectively). mtMutS1 bound to mismatched DNAs with a higher affinity than to perfectly-matched and other structured DNAs, which resembles the DNA-binding specificities of eukaryotic and bacterial MutS1 homologs. The mtMutL CTD showed a Mn2+/Ni2+/Co2+-dependent nicking endonuclease activity that introduces single-strand breaks into a circular double-stranded DNA. The nicking endonuclease activity of the mtMutL CTD was impaired by mutagenizing the metal-binding motif that is identical to those of eukaryotic and bacterial MutL endonucleases. These results raise the possibility that not only the EndoMS-dependent MMR but also the traditional MutS1/MutL-dependent MMR exist in archaea.
Subject(s)
Methanosarcinales/enzymology , MutL Proteins/metabolism , Amino Acid Sequence , DNA Mismatch Repair , Methanosarcinales/metabolism , Models, Molecular , MutL Proteins/chemistry , Proliferating Cell Nuclear Antigen/metabolism , Protein Multimerization , Protein Structure, QuaternaryABSTRACT
In plant organelle genomes, homeologous recombination between heteroallelic positions of repetitive sequences is increased by dysfunction of the gene encoding MutS homolog 1 (MSH1), a plant organelle-specific homolog of bacterial mismatch-binding protein MutS1. The C-terminal region of plant MSH1 contains the GIY-YIG endonuclease motif. The biochemical characteristics of plant MSH1 have not been investigated; accordingly, the molecular mechanism by which plant MSH1 suppresses homeologous recombination is unknown. Here, we characterized the recombinant GIY-YIG domain of Arabidopsis thaliana MSH1, showing that the domain possesses branched DNA-specific DNA-binding activity. Interestingly, the domain exhibited no endonuclease activity, suggesting that the mismatch-binding domain is required for DNA incision. Based on these results, we propose a possible mechanism for MSH1-dependent suppression of homeologous recombination.
Subject(s)
Arabidopsis Proteins/genetics , DNA/metabolism , Endonucleases/genetics , MutS DNA Mismatch-Binding Protein/genetics , Amino Acid Sequence , Arabidopsis Proteins/metabolism , Binding Sites/genetics , DNA/genetics , Endonucleases/classification , Endonucleases/metabolism , Models, Genetic , MutS DNA Mismatch-Binding Protein/metabolism , Phylogeny , Recombinational DNA Repair , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutSγ, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon Pyrococcus horikoshii (phMutS5). Archaeal MutS5 has a Walker ATPase motif-containing amino acid sequence that shows similarity to the ATPase domain of MutSγ. It is known that the ATPase domain of MutS homologs is also a dimerization domain. Chemical cross-linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear DNAs, which resembles the DNA-binding specificities of MutSγ and bacterial MutS2, a Holliday junction-resolving MutS homolog. However, phMutS5 has no nuclease activity against branched DNA unlike MutS2. The ATPase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutSγ. Furthermore, site-directed mutagenesis revealed that the ATPase activity is dependent on the Walker ATPase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutSγ.
