MutS homologues are highly conserved enzymes engaged in DNA mismatch repair (MMR), meiotic recombination and other DNA modifications. Genome sequencing projects have revealed that bacteria and plants possess a MutS homologue, MutS2. MutS2 lacks the mismatch-recognition domain of MutS, but contains an extra C-terminal region called the small MutS-related (Smr) domain. Sequences homologous to the Smr domain are annotated as ‘proteins of unknown function’ in various organisms ranging from bacteria to human. Although recent in vivo studies indicate that MutS2 plays an important role in recombinational events, there had been only limited characterization of the biochemical function of MutS2 and the Smr domain. We previously established that Thermus thermophilus MutS2 (ttMutS2) possesses endonuclease activity. In this study, we report that a Smr-deleted ttMutS2 mutant retains the dimerization, ATPase and DNA-binding activities, but has no endonuclease activity. Furthermore, the Smr domain alone was stable and functional in binding and incising DNA. It is noteworthy that an endonuclease activity is associated with a MutS homologue, which is generally thought to recognize specific DNA structures.
In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of Escherichia coli. A mutS::aac mutant showed 20- to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS+. The mutS+ gene of strain ATCC 17587 did not complement an E. coli mutant but partially complemented a P. stutzeri JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of mutS+ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in P. stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.
The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.
Mismatch repair (MMR) is an evolutionarily conserved DNA repair system, which corrects mismatched bases arising during DNA replication. MutS recognizes and binds base pair mismatches, while the MutL protein interacts with MutS–mismatch complex and triggers MutH endonuclease activity at a distal-strand discrimination site on the DNA. The mechanism of communication between these two distal sites on the DNA is not known. We used functional fluorescent MMR proteins, MutS and MutL, in order to investigate the formation of the fluorescent MMR protein complexes on mismatches in real-time in growing Escherichia coli cells. We found that MutS and MutL proteins co-localize on unrepaired mismatches to form fluorescent foci. MutL foci were, on average, 2.7 times more intense than the MutS foci co-localized on individual mismatches. A steric block on the DNA provided by the MutHE56A mutant protein, which binds to but does not cut the DNA at the strand discrimination site, decreased MutL foci fluorescence 3-fold. This indicates that MutL accumulates from the mismatch site toward strand discrimination site along the DNA. Our results corroborate the hypothesis postulating that MutL accumulation assures the coordination of the MMR activities between the mismatch and the strand discrimination site.
The MutS protein of Escherichia coli plays a key role in the recognition and repair of errors made during the replication of DNA. Homologs of MutS have been found in many species including eukaryotes, Archaea and other bacteria, and together these proteins have been grouped into the MutS family. Although many of these proteins have similar activities to the E.coli MutS, there is significant diversity of function among the MutS family members. This diversity is even seen within species; many species encode multiple MutS homologs with distinct functions. To better characterize the MutS protein family, I have used a combination of phylogenetic reconstructions and analysis of complete genome sequences. This phylogenomic analysis is used to infer the evolutionary relationships among the MutS family members and to divide the family into subfamilies of orthologs. Analysis of the distribution of these orthologs in particular species and examination of the relationships within and between subfamilies is used to identify likely evolutionary events (e.g. gene duplications, lateral transfer and gene loss) in the history of the MutS family. In particular, evidence is presented that a gene duplication early in the evolution of life resulted in two main MutS lineages, one including proteins known to function in mismatch repair and the other including proteins known to function in chromosome segregation and crossing-over. The inferred evolutionary history of the MutS family is used to make predictions about some of the uncharacterized genes and species included in the analysis. For example, since function is generally conserved within subfamilies and lineages, it is proposed that the function of uncharacterized proteins can be predicted by their position in the MutS family tree. The uses of phylogenomic approaches to the study of genes and genomes are discussed.
Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.
Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2–Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30–50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1ADP–S2ATP is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1ATP–S2ATP and S1ATP–S2ADP are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair.
Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLα, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH2 termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLα, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLα demonstrated that the NH2 terminus of MutLα undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH2 termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLα undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of damaged base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between MutS and the effects of methylating versus ethylating agents on mutagenesis, using a MutS deficient strain of E. coli. We found that methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, whereas the genetic background did not affect mutagenicity of ethylating agents. Thus, the role of the MMR system in repair with methylation-induced mutagenesis appears to be greater than its role with ethylation-induced mutagenesis. To further understand E. coli MutS, we compared binding of the protein to O6-alkylated guanine paired with thymine, which could lead to mutation, versus cytosine, which cannot. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O6-MeG:T, O6-MeG:C, O6-EtG:T, O6-EtG:C, G:T and G:C. The dissociation constants (Kd's), which reflect the strength of binding, followed the order G:T- > O6-MeG:T- > O6-EtG:T- = O6-EtG:C- ≥ O6-MeG:C- > G:C. These results suggest that a thymine paired with O6-methylated guanine is recognized as a mismatch by MutS and removed more efficiently than a thymine opposite O6-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induces lesions than those caused by ethylation.
MutS; mismatch repair; O6-methylguanine; O6-ethylguanine; mutation
DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired DNA nicking and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.
DNA mismatch repair; MutL; endonuclease; HNPCC; β-clamp
MutS homologs, identified in nearly all bacteria and eukaryotes, include the bacterial proteins MutS1 and MutS2 and the eukaryotic MutS homologs 1 to 7, and they often are involved in recognition and repair of mismatched bases and small insertion/deletions, thereby limiting illegitimate recombination and spontaneous mutation. To explore the relationship of MutS2 to other MutS homologs, we examined conserved protein domains. Fundamental differences in structure between MutS2 and other MutS homologs suggest that MutS1 and MutS2 diverged early during evolution, with all eukaryotic homologs arising from a MutS1 ancestor. Data from MutS1 crystal structures, biochemical results from MutS2 analyses, and our phylogenetic studies suggest that MutS2 has functions distinct from other members of the MutS family. A mutS2 mutant was constructed in Helicobacter pylori, which lacks mutS1 and mismatch repair genes mutL and mutH. We show that MutS2 plays no role in mismatch or recombinational repair or deletion between direct DNA repeats. In contrast, MutS2 plays a significant role in limiting intergenomic recombination across a range of donor DNA tested. This phenotypic analysis is consistent with the phylogenetic and biochemical data suggesting that MutS1 and MutS2 have divergent functions.
MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLα (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov et al. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLα, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLα is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSα, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLα endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLα ATPase activity or the ability of the protein to support assembly of the yMutLα•yMutSα•heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLα incision to the discontinuous strand of a nicked heteroduplex.
The DNA mismatch repair protein MutS acts as a molecular switch. It toggles between ADP and ATP states and is regulated by mismatched DNA. This is analogous to G-protein switches and the regulation of their “on” and “off” states by guanine exchange factors. Although GDP release in monomeric GTPases is accelerated by guanine exchange factor-induced removal of magnesium from the catalytic site, we found that release of ADP from MutS is not influenced by the metal ion in this manner. Rather, ADP release is induced by the binding of mismatched DNA at the opposite end of the protein, a long-range allosteric response resembling the mechanism of activation of heterotrimeric GTPases. Magnesium influences switching in MutS by inducing faster and tighter ATP binding, allowing rapid downstream responses. MutS mutants with decreased affinity for the metal ion are impaired in fast switching and in vivo mismatch repair. Thus, the G-proteins and MutS conceptually employ the same efficient use of the high energy cofactor: slow hydrolysis in the absence of a signal and fast conversion to the active state when required.
DNA/Protein Interaction; DNA/Repair; Enzymes/ATPases; Enzymes/Mechanisms; Enzymes/Structure; G-proteins; Protein/Metal Ion Interaction; Protein/Structure; DNA mismatch repair; Magnesium
Escherichia coli MutY and MutS increase replication fidelity by removing adenines that were misincorporated opposite 7,8-dihydro-8-oxo-deoxyguanines (8-oxoG), G, or C. MutY DNA glycosylase removes adenines from these mismatches through a short-patch base excision repair pathway and thus prevents G:C-to-T:A and A:T-to-G:C mutations. MutS binds to the mismatches and initiates the long-patch mismatch repair on daughter DNA strands. We have previously reported that the human MutY homolog (hMYH) physically and functionally interacts with the human MutS homolog, hMutSα (Y. Gu et al., J. Biol. Chem. 277:11135-11142, 2002). Here, we show that a similar relationship between MutY and MutS exists in E. coli. The interaction of MutY and MutS involves the Fe-S domain of MutY and the ATPase domain of MutS. MutS, in eightfold molar excess over MutY, can enhance the binding activity of MutY with an A/8-oxoG mismatch by eightfold. The MutY expression level and activity in mutS mutant strains are sixfold and twofold greater, respectively, than those for the wild-type cells. The frequency of A:T-to-G:C mutations is reduced by two- to threefold in a mutS mutY mutant compared to a mutS mutant. Our results suggest that MutY base excision repair and mismatch repair defend against the mutagenic effect of 8-oxoG lesions in a cooperative manner.
Mismatch repair corrects errors that have escaped polymerase proofreading enhancing replication fidelity by at least two orders of magnitude. The β and PCNA sliding clamps increase the polymerase processivity during DNA replication and are important at several stages of mismatch repair. Both MutS and MutL, the two proteins that initiate the mismatch repair response, interact with β. Binding of MutS to β is important to recruit MutS and MutL to foci. Moreover, the endonuclease activity of human and yeast MutLα is stimulated by PCNA. However, the concrete functions of the processivity clamp in the repair steps preceding DNA resynthesis remain obscure. Here, we demonstrate that the C-terminal domain of MutL encompasses a bona fide β-binding motif that mediates a weak, yet specific, interaction between the two proteins. Mutation of this conserved motif correlates with defects in mismatch repair, demonstrating that the direct interaction with β is important for MutL function. The interaction between the C-terminal domain of MutL and β is conserved in both B. subtilis and E. coli, but the repair defects associated with mutation of this β-binding motif are more severe in the former, suggesting that this interaction may have a more prominent role in methyl-independent than methyl-directed mismatch repair systems. Together with previously published data, our work strongly suggests that β may stimulate the endonuclease activity of MutL through its direct interaction with the C-terminal domain of MutL.
DNA mismatch repair; MutL; β-sliding clamp; PCNA; endonuclease
The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.
To understand the evolutionary process of the DNA mismatch repair system, we conducted systematic phylogenetic analysis of its key components, the bacterial MutS and MutL genes and their eukaryotic homologs. Based on genome-wide homolog searches, we identified three new MutS subfamilies (MutS3-5) in addition to the previously studied MutS1 and MutS2 subfamilies. Detailed evolutionary analysis strongly suggests that frequent ancient horizontal gene transfer (HGT) occurred with both MutS and MutL genes from bacteria to eukaryotes and/or archaea. Our results further imply that the origins of mismatch repair system in eukaryotes and archaea are largely attributed to ancient HGT from bacteria instead of vertical evolution. Specifically, the eukaryotic MutS and MutL homologs likely originated from endosymbiotic ancestors of mitochondria or chloroplasts, indicating that not only archaea, but also bacteria are important sources of eukaryotic DNA metabolic genes. The archaeal MutS1 and MutL homologs were also acquired from bacteria simultaneously through HGT. Moreover, the distribution and evolution profiles of the MutS1 and MutL genes suggest that they have undergone long-term coevolution. Our work presents an overall portrait of the evolution of these important genes in DNA metabolism and also provides further understanding about the early evolution of cellular organisms.
The DNA binding properties of hMutSα and hMutLα and complex formation of hMutSα with hMutLα and hMutLβ were investigated using binding experiments on magnetic bead-coupled DNA substrates with nuclear extracts as well as purified proteins. hMutSα binding to homoduplex DNA was disrupted by lower NaCl concentrations than hMutSα binding to a mismatch. ATP markedly reduced the salt resistance of hMutSα binding but hMutSα still retained affinity for heteroduplexes. hMutSα formed a complex with hMutLα and hMutLβ on DNA in the presence of ATP. This complex only formed on 81mer and not 32mer DNA substrates. Complex formation was enhanced by a mismatch in the DNA substrate, and hMutLα and hMutLβ were shown to enter the complex at different ATP concentrations. Purified hMutLα showed an intrinsic affinity for DNA, with a preference for single-stranded over double-stranded DNA.
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O6-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O6-MeG:T, O6-MeG:C, O6-EtG:T, O6-EtG:C, G:T and G:C. Dissociation constants (Kd), which reflect the strength of binding, followed the order G:T- > O6-MeG:T- > O6-EtG:T- = O6-EtG:C- ≥ O6-MeG:C- > G:C. These results suggest that a thymine base paired with O6-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O6-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.
MutS; Mismatch repair; O6-methylguanine; O6-ethylguanine; Mutation
MutS protein binds to DNA and specifically recognizes mismatched or small looped out heteroduplex DNA. In order to elucidate its structure-function relationships, the domain structure of Thermus thermophilus MutS protein was studied by performing denaturation experiments and limited proteolysis. The former suggested that T. thermophilus MutS consists of at least three domains with estimated stabilities of 12.3, 22.9 and 30.7 kcal/mol and the latter revealed that it consists of four domains: A1 (N-terminus to residue 130), A2 (131-274), B (275-570) and C (571 to C-terminus). A gel retardation assay indicated that T.thermophilus MutS interacts non-specifically with double-stranded (ds), but not single-stranded DNA. Among the proteolytic fragments, the B domain bound to dsDNA. On the basis of these results we have proposed the domain organization of T. thermophilus MutS and putative roles of these domains.
DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch Syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutSα is well characterized. We report here crystal structures of human MutSβ complexed with DNA containing insertion-deletion loops (IDL) of 2, 3, 4, or 6 unpaired nucleotides. In contrast to eukaryotic MutSα and bacterial MutS, which bind the base of a mismatched nucleotide, MutSβ binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flipped out into the major groove and partially exposed to solvent. A normal downstream basepair can become unpaired; thereby a single unpaired base can be converted to an IDL of 2 nucleotides and recognized by MutSβ. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hydrolysis by Msh2 and Msh3 with mismatch binding to signal for repair.
mismatch repair; asymmetric dimer; Msh3; ATPase; trinucleotide repeat
Most eubacteria, and all eukaryotes examined thus far, encode homologs of the DNA mismatch repair protein MutS. Although eubacteria encode only one or two MutS-like proteins, eukaryotes encode at least six distinct MutS homolog (MSH) proteins, corresponding to conserved (orthologous) gene families. This suggests evolution of individual gene family lines of descent by several duplication/specialization events. Using quantitative phylogenetic analyses (RASA, or relative apparent synapomorphy analysis), we demonstrate that comparison of complete MutS protein sequences, rather than highly conserved C-terminal domains only, maximizes information about evolutionary relationships. We identify a novel, highly conserved middle domain, as well as clearly delineate an N-terminal domain, previously implicated in mismatch recognition, that shows family-specific patterns of aromatic and charged amino acids. Our final analysis, in contrast to previous analyses of MutS-like sequences, yields a stable phylogenetic tree consistent with the known biochemical functions of MutS/MSH proteins, that now assigns all known eukaryotic MSH proteins to a monophyletic group, whose branches correspond to the respective specialized gene families. The rooted phylogenetic tree suggests their derivation from a mitochondrial MSH1-like protein, itself the descendent of the MutS of a symbiont in a primitive eukaryotic precursor.
We have examined interaction parameters, conformation, and functional significance of the human MutSα·PCNA complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a KD of 0.7 μM in absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSα for a mismatch, and mismatch-bound MutSα binds PCNA. Small angle X-ray scattering studies have established molecular parameters of the complex and are consistent with an elongated conformation in which the two proteins associate in end-to-end fashion in a manner that does not involve an extended unstructured tether as has been proposed for yeast MutSα and PCNA (Shell et al. (2007)
Mol. Cell 26, 565-578). MutSα variants lacking the PCNA interaction motif are functional in 3′- or 5′-directed mismatch-provoked excision, but display a partial defect in 5′-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSα PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in mismatch repair.
Vsr DNA mismatch endonuclease is the key enzyme of very short patch (VSP) DNA mismatch repair and nicks the T-containing strand at the site of a T-G mismatch in a sequence-dependent manner. MutS is part of the mutHLS repair system and binds to diverse mismatches in DNA. The function of the mutL gene product is currently unclear but mutations in the gene abolish mutHLS -dependent repair. The absence of MutL severely reduces VSP repair but does not abolish it. Purified MutL appears to act catalytically to bind Vsr to its substrate; one-hundredth of an equivalent of MutL is sufficient to bring about a significant effect. MutL enhances binding of MutS to its substrate 6-fold but does so in a stoichiometric manner. Mutational studies indicate that the MutL interaction region lies within the N-terminal 330 amino acids and that the MutL multimerization region is at the C-terminal end. MutL mutant monomeric forms can stimulate MutS binding.
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.
mimivirus; girus; virus; DNA repair; MutS