Crystal structure of MutS2 endonuclease domain and the mechanism of homologous recombination suppression

DNA recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. Recent studies revealed the existence of a novel anti-recombination enzyme, MutS2. However, the mechanism by which MutS2 inhibits homologous recombination has been unknown. Previously, we found that Thermus thermophilus MutS2 (ttMutS2) harbors an endonuclease activity and that this activity is confined to the C-terminal domain, whose amino acid sequence is widely conserved in a variety of proteins with unknown function from almost all organisms ranging from bacteria to man. In this study, we determined the crystal structure of the ttMutS2 endonuclease domain at 1.7-angstroms resolution, which resembles the structure of the DNase I-like catalytic domain of Escherichia coli RNase E, a sequence-nonspecific endonuclease. The N-terminal domain of ttMutS2, however, recognized branched DNA structures, including the Holliday junction and D-loop structure, a primary intermediate in homologous recombination. The full-length of ttMutS2 digested the branched DNA structures at the junction. These results indicate that ttMutS2 suppresses homologous recombination through a novel mechanism involving resolution of early intermediates.     

Thermus thermophilus MutS2, a MutS paralogue, possesses an endonuclease activity promoted by MutL

The mismatch repair system (MMR) recognizes and corrects mismatched or unpaired bases caused mainly by DNA polymerase, and contributes to the fidelity of DNA replication in living cells. In Escherichia coli, the MutHLS system is known to function in MMR, and homologues of MutS and MutL are widely conserved in almost all organisms. However, the MutH endonuclease has not been found in the majority of organisms. Such organisms, including Thermus thermophilus HB8, often possess the so-called MutS2 protein, which is highly homologous to MutS but contains an extra C-terminal stretch. To elucidate the function of MutS2, we overexpressed and purified T. thermophilus MutS2 (ttMutS2). ttMutS2 demonstrated the ability to bind double-stranded (ds) DNA, but, unlike ttMutS, ttMutS2 showed no specificity for mismatched duplexes. ttMutS2 ATPase activity was also detected and was stimulated by dsDNA. Our results also showed that ttMutS2 incises dsDNA. ttMutS2 incises not only oligo dsDNA but also plasmid DNA, suggesting that ttMutS2 possesses an endonuclease activity. At low concentrations, the incision activity was not retained, but was promoted by T. thermophilus MutL.     

Biochemical characterization of mismatch-binding protein MutS1 and nicking endonuclease MutL from a euryarchaeon Methanosaeta thermophila

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.     

Analysis of a nuclease activity of catalytic domain of Thermus thermophilus MutS2 by high-accuracy mass spectrometry

Electrospray ionization with Fourier-transform ion cyclotron resonance mass spectrometry (ESI–FT ICR MS) is a powerful tool for analyzing the precise structural features of biopolymers, including oligonucleotides. Here, we described the detailed characterization of a newly discovered nuclease activity of the C-terminal domain of Thermus thermophilus MutS2 (ttMutS2). Using this method, the length, nucleotide content and nature of the 5′- and 3′-termini of the product oligonucleotides were accurately identified. It is revealed that the C-terminal domain of ttMutS2 incised the phosphate backbone of oligodeoxynucleotides non-sequence-specifically at the 3′ side of the phosphates. The simultaneous identification of the innumerable fragments was achieved by the extremely high-accuracy of ESI–FT ICR MS.     

Structural and functional studies of MutS2 from Deinococcus radiodurans

The MutS2 homologues have been found widespread in most prokaryotes, which are involved in DNA repair and reactive oxygen species detoxification. The C-terminal small mutS-related (Smr) domain is critical for its endonucleolytic activity. However, the detailed catalytic mechanism is still unclear. In this study, we first investigated the in vivo role of drMutS2 in Deinococcus radiodurans, the most radiation-resistant organism exhibits the remarkable DNA repair capacity. mutS2 and recA mutS2 double knockout mutants were constructed because the phenotype was strongly masked by the predominant homologous recombination DNA repair pathway in this bacterium. Compared with the recA mutant, cells devoid of both genes showed increased sensitivity to ionizing radiation and oxidative agents, suggesting that drMutS2 is involved in RecA-independent mechanisms that enhance cellular resistance to oxidative stress-induced DNA damage. Moreover, the basal level of reductase activity and thiamine biosynthesis was induced in the absence of mutS2. To characterize its catalytic residues, the Smr domain was crystallized and soaked in buffer containing manganese ions. In contrast to native crystals, the space group of manganese-derivative crystals transformed from monoclinic to orthorhombic unexpectedly. This type of crystals showed improved diffraction resolution to 1.2 Å, which has the highest resolution of currently known Smr structures. Structural comparison revealed that three acidic amino-acid residues, which are all located in the α1 helix, changed the rotamer states after metal soaking. Mutational analysis of conserved residue glutamic acid 710 to alanine yielded a drMutS2 variant with impaired nuclease activity, and could only partially rescue the radiosensitive phenotype of the mutS2 null strain, indicating that glutamic acid 710 is the catalytic residue.     

The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti‐recombination function of Helicobacter pylori MutS2

Helicobacter pylori, a human pathogen, is a naturally and constitutively competent bacteria, displaying a high rate of intergenomic recombination. While recombination events are essential for evolution and adaptation of H. pylori to dynamic gastric niches and new hosts, such events should be regulated tightly to maintain genomic integrity. Here, we analyze the role of the nuclease activity of MutS2, a protein that limits recombination during transformation in H. pylori. In previously studied MutS2 proteins, the C‐terminal Smr domain was mapped as the region responsible for its nuclease activity. We report here that deletion of Smr domain does not completely abolish the nuclease activity of HpMutS2. Using bioinformatics analysis and mutagenesis, we identified an additional and novel nuclease motif (LDLK) at the N‐terminus of HpMutS2 unique to Helicobacter and related ε‐proteobacterial species. A single point mutation (D30A) in the LDLK motif and the deletion of Smr domain resulted in ～ 5–10‐fold loss of DNA cleavage ability of HpMutS2. Interestingly, the mutant forms of HpMutS2 wherein the LDLK motif was mutated or the Smr domain was deleted were unable to complement the hyper‐recombination phenotype of a mutS2^? strain, suggesting that both nuclease sites are indispensable for an efficient anti‐recombinase activity of HpMutS2.     

Crystal structure of YdaL, a stand-alone small MutS-related protein from Escherichia coli

Sequence homologs of the small MutS-related (Smr) domain, the C-terminal endonuclease domain of MutS2, also exist as stand-alone proteins. In this study, we report the crystal structure of a proteolyzed fragment of YdaL (YdaL??-???), a stand-alone Smr protein from Escherichia coli. In this structure, residues 86-170 assemble into a classical Smr core domain and are embraced by an N-terminal extension (residues 40-85) with an α/β/α fold. Sequence alignment indicates that the N-terminal extension is conserved among a number of stand-alone Smr proteins, suggesting structural diversity among Smr domains. We also discovered that the DNA binding affinity and endonuclease activity of the truncated YdaL??-??? protein were slightly lower than those of full-length YdaL?-???, suggesting that residues 1-38 may be involved in DNA binding.     

Structural and functional analysis of the MutS C-terminal tetramerization domain

The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801–853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an α-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutS^CF/D835R is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1–800), which lacks the CTD. Moreover, the dimer-forming MutS^CF/D835R has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.     

Solution structure and characterization of the DNA-binding activity of the B3BP-Smr domain

The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer α-β sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.     

Two new subfamilies of DNA mismatch repair proteins (MutS) specifically abundant in the marine environment

MutS proteins are ubiquitous in cellular organisms and have important roles in DNA mismatch repair or recombination. In the virus world, the amoeba-infecting Mimivirus, as well as the recently sequenced Cafeteria roenbergensis virus are known to encode a MutS related to the homologs found in octocorals and ɛ-proteobacteria. To explore the presence of MutS proteins in other viral genomes, we performed a genomic survey of four giant viruses (‘giruses'') (Pyramimonas orientalis virus (PoV), Phaeocystis pouchetii virus (PpV), Chrysochromulina ericina virus (CeV) and Heterocapsa circularisquama DNA virus (HcDNAV)) that infect unicellular marine algae. Our analysis revealed the presence of a close homolog of Mimivirus MutS in all the analyzed giruses. These viral homologs possess a specific domain structure, including a C-terminal HNH-endonuclease domain, defining the new MutS7 subfamily. We confirmed the presence of conserved mismatch recognition residues in all members of the MutS7 subfamily, suggesting their role in DNA mismatch repair rather than DNA recombination. PoV and PpV were found to contain an additional type of MutS, which we propose to call MutS8. The MutS8 proteins in PoV and PpV were found to be closely related to homologs from ‘Candidatus Amoebophilus asiaticus'', an obligate intracellular amoeba-symbiont belonging to the Bacteroidetes. Furthermore, our analysis revealed that MutS7 and MutS8 are abundant in marine microbial metagenomes and that a vast majority of these environmental sequences are likely of girus origin. Giruses thus seem to represent a major source of the underexplored diversity of the MutS family in the microbial world.     

MutS inhibits RecA-mediated strand transfer with methylated DNA substrates

DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O⁶-methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O⁶-methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecA-mediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentration-dependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O⁶-methylguanine residues in methylated DNA by HPLC-MS/MS and 5–10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system.     

One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA

A novel, universal method for mutation detection utilising the ability of MutS protein to recognise DNA incomplementarities is proposed. The examined and reference DNA fragments are PCR amplified. The PCR products are purified, mixed, heated and cooled to form heteroduplexes. In the case of mutation the heteroduplex DNA containing mismatch is protected against exonuclease digestion by MutS, while the DNA without mismatches is degraded. The protection effect is visualised by the direct addition of a highly sensitive fluorescent dye (SYBR-Gold) selectively binding DNA. The Thermus thermophilus recombined His-tagged MutS and 3′–5′ exonuclease activity of T4 DNA polymerase were used in the assay.     

Vanadate inhibits the ATPase activity and DNA binding capability of bacterial MutS. A structural model for the vanadate-MutS interaction at the Walker A motif

MutS, a member of the ABC ATPases superfamily, is a mismatch DNA-binding protein constituent of the DNA post-replicative mismatch repair system (MMRS). In this work, it is shown that the ATPase activity of Pseudomonas aeruginosa and Escherichia coli MutS is inhibited by ortho- and decavanadate. Structural comparison of the region involved in the ATP binding of E.coli MutS with the corresponding region of other ABC ATPases inhibited by vanadate, including the myosin– orthovanadate–Mg complex, showed that they are highly similar. From these results it is proposed that the orthovanadate inhibition of MutS ATPase can take place by a similar mechanism to that described for other ATPases. Docking of decavanadate on the ATP-binding region of MutS showed that the energetically more favorable interaction of this compound would take place with the complex MutS– ADP–Mg, suggesting that the inhibitory effect could be produced by a steric impediment of the protein ATP/ADP exchange. Besides the effect observed on the ATPase activity, vanadate also affects the DNA-binding capability of the protein, and partially inhibits the oligomerization of MutS and the temperature-induced inactivation of the protein. From the results obtained, and considering that vanadate is an intracellular trace component, this compound could be considered as a new modulator of the MMRS.     

Dual recognition-incision enzymes might be involved in mismatch repair and meiosis

Mismatch repair in many organisms depends on three proteins: the mismatch-recognition protein MutS, a nicking endonuclease MutH, and MutL, which acts as a scaffold between these. However, many genomes lack MutL but possess MutS. In one of these cases, in a coral mitochondrial genome, a gene is present that encodes a MutS protein fused to an HNH nicking endonuclease, potentially eliminating the requirement for MutL. Likewise, many prokaryotes could operate similarly, independently of MutL by encoding a fused MutS-Smr (MutS2) protein. Smr, which is proposed to be a nicking endonuclease, can also be found separately in many eukaryotes, where it might play a role in mismatch repair or meiotic chromosome crossing-over.     

Steady-state ATPase activity of E. coli MutS modulated by its dissociation from heteroduplex DNA

The ability of MutS to recognize mismatched DNA is required to initiate a mismatch repair (MMR) system. ATP binding and hydrolysis are essential in this process, but their role in MMR is still not fully understood. In this study, steady-state ATPase activities of MutS from Escherichia coli were investigated using the spectrophotometric method with a double end-blocked heteroduplex containing gapped bases. The ATPase activities of MutS increased as the number of gapped bases increased in a double end-blocked heteroduplex with 2-8 gapped bases in the chain, indicating that MutS dissociates from DNA when it reaches a scission during movement along the DNA. Since movement of MutS along the chain does not require extensive ATP hydrolysis and the ATPase activity is only enhanced when MutS dissociates from a heteroduplex, these results support the sliding clamp model in which ATP binding by MutS induces the formation of a hydrolysis-independent sliding clamp.     

Spatial coupling between DNA replication and mismatch repair in Caulobacter crescentus

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.     

Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex

The roles of ATP binding and hydrolysis by MutS in mismatch repair are poorly understood. MutS E694A, in which Glu-694 of the Walker B motif is substituted with alanine, is defective in hydrolysis of bound ATP and has been reported to support MutL-dependent activation of the MutH d(GATC) endonuclease in a trans DNA activation assay (Junop, M. S., Obmolova, G., Rausch, K., Hsieh, P., and Yang, W. (2001) Mol. Cell 7, 1-12). Because the MutH trans activation assay used in these previous studies was characterized by high background and low efficiency, we have re-evaluated the activities of MutS E694A. In contrast to native MutS, which can be isolated in a nucleotide-free form, purified MutS E694A contains 1.0 mol of bound ATP per dimer equivalent, and substoichiometric levels of bound ADP (0.08-0.58 mol/dimer), consistent with the suggestion that the ADP.MutS.ATP complex comprises a significant fraction of the protein in solution (Bjornson, K. P. and Modrich, P. (2003) J. Biol. Chem. 278, 18557-18562). In the presence of Mg2+, endogenous ATP is hydrolyzed with a rate constant of 0.12 min-1 at 30 degrees C, and hydrolysis yields a protein that displays increased specificity for heteroduplex DNA. As observed with wild type MutS, ATP can promote release of MutS E694A from a mismatch. However, the mutant protein is defective in the methyl-directed, mismatch- and MutL-dependent cis activation of MutH endonuclease on a 6.4-kilobase pair heteroduplex, displaying only 1 to 2% of the activity of wild type MutS. The mutant protein also fails to support normal assembly of the MutS.MutL.DNA ternary complex. Although a putative ternary complex can be observed in the presence of MutS E694A, assembly of this structure displays little if any dependence on a mismatched base pair.     

Suppression of homologous and homeologous recombination by the bacterial MutS2 protein

In addition to their role in DNA repair, recombination events are associated with processes aimed at providing the genetic variability needed for adaptation and evolution of a population. In bacteria, recombination is involved in the appearance of new variants by allowing the incorporation of exogenous DNA or the reshuffling of endogenous sequences. Here we show that HpMutS2, a protein belonging to the MutS2 family in Helicobacter pylori, is not involved in mismatch repair but inhibits homologous and homeologous recombination. Disruption of HpmutS2 leads to an increased efficiency of exogenous DNA incorporation. HpMutS2 has a selective affinity for DNA structures mimicking recombination intermediates with no specificity for homoduplex DNA or mismatches. The purified protein has an ATPase activity stimulated by the same DNA structures. Finally, we show that HpMutS2 inhibits DNA strand exchange reactions in vitro. Thus, MutS2 proteins are candidates for controlling recombination and therefore genetic diversity in bacteria.     

The role of MutS oligomers on Pseudomonas aeruginosa mismatch repair system activity

The Escherichia coli DNA Mismatch Repair (MMR) protein MutS exist as dimers and tetramers in solution, and the identification of its functional oligomeric state has been matter of extensive study. In the present work, we have analyzed the oligomerization state of MutS from Pseudomonas aeruginosa a bacterial species devoid of Dam methylation and MutH homologue. By analyzing native MutS and different mutated versions of the protein, we determined that P. aeruginosa MutS is mainly tetrameric in solution and that its oligomerization capacity is conducted as in E. coli, by the C-terminal region of the protein. The analysis of mismatch oligonucleotide binding activity showed that wild-type MutS binds to DNA as tetramer. The DNA binding activity decreased when the C-terminal region was deleted (MutSDelta798) or when a full-length MutS with tetramerization defects (MutSR842E) was tested. The ATPase activity of MutSDelta798 was similar to MutSR842E and diminished respect to the wild-type protein. Experiments carried out on a P. aeruginosa mutS strain to test the proficiency of different oligomeric versions of MutS to function in vivo showed that MutSDelta798 is not functional and that full-length dimeric version MutSR842E, is not capable of completely restoring the MMR activity of the mutant strain. Additional experiments carried out in conditions of high mutation rate induced by the base analogue 2-AP confirm that the dimeric version of MutS is not as efficient as the tetrameric wild-type protein to prevent mutations. Therefore, it is concluded that although dimeric MutS is sufficient for MMR activity, optimal activity is obtained with the tetrameric version of the protein and therefore it should be considered as the active form of MutS in P. aeruginosa.     

Different Roles of Eukaryotic MutS and MutL Complexes in Repair of Small Insertion and Deletion Loops in Yeast

DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair.