Structural basis for MutH activation in E.coli mismatch repair and relationship of MutH to restriction endonucleases.

MutS, MutL and MutH are the three essential proteins for initiation of methyl-directed DNA mismatch repair to correct mistakes made during DNA replication in Escherichia coli. MutH cleaves a newly synthesized and unmethylated daughter strand 5' to the sequence d(GATC) in a hemi-methylated duplex. Activation of MutH requires the recognition of a DNA mismatch by MutS and MutL. We have crystallized MutH in two space groups and solved the structures at 1.7 and 2.3 A resolution, respectively. The active site of MutH is located at an interface between two subdomains that pivot relative to one another, as revealed by comparison of the crystal structures, and this presumably regulates the nuclease activity. The relative motion of the two subdomains in MutH correlates with the position of a protruding C-terminal helix. This helix appears to act as a molecular lever through which MutS and MutL may communicate the detection of a DNA mismatch and activate MutH. With sequence homology to Sau3AI and structural similarity to PvuII endonuclease, MutH is clearly related to these enzymes by divergent evolution, and this suggests that type II restriction endonucleases evolved from a common ancestor.     

Interaction of Escherichia coli MutS and MutL at a DNA mismatch

MutS and MutL are both required to activate downstream events in DNA mismatch repair. We examined the rate of dissociation of MutS from a mismatch using linear heteroduplex DNAs or heteroduplexes blocked at one or both ends by four-way DNA junctions in the presence and absence of MutL. In the presence of ATP, dissociation of MutS from linear heteroduplexes or heteroduplexes blocked at only one end occurs within 15 s. When both duplex ends are blocked, MutS remains associated with the DNA in complexes with half-lives of 30 min. DNase I footprinting of MutS complexes is consistent with migration of MutS throughout the DNA duplex region. When MutL is present, it associates with MutS and prevents ATP-dependent migration away from the mismatch in a manner that is dependent on the length of the heteroduplex. The rate and extent of mismatch-provoked cleavage at hemimethylated GATC sites by MutH in the presence of MutS, MutL, and ATP are the same whether the mismatch and GATC sites are in cis or in trans. These results suggest that a MutS-MutL complex in the vicinity of a mismatch is involved in activating MutH.     

DNA mismatch correction in Haemophilus influenzae: characterization of MutL, MutH and their interaction

Haemophilus influenzae DNA mismatch repair proteins, MutS, MutL and MutH, are functionally characterized in this study. Introduction of mutS, mutL and mutH genes of H. influenzae resulted in complementation of the mismatch repair activity of the respective mutant strains of Escherichia coli to varying levels. DNA binding studies using H. influenzae MutH have shown that the protein is capable of binding to any DNA sequence non-specifically in a co-operative and metal independent manner. Presence of MutL and ATP in the binding reaction resulted in the formation of a more specific complex, which indicates that MutH is conferred specificity for binding hemi-methylated DNA through structural alterations mediated by its interaction with MutL. To study the role of conserved amino acids Ile213 and Leu214 in the helix at the C-terminus of MutH, they were mutated to alanine. The mutant proteins showed considerably reduced DNA binding and nicking, as well as MutL-mediated activation. MutH failed to nick HU bound DNA whereas MboI and Sau3AI, which have the same recognition sequence as MutH, efficiently cleaved the substrate. MutS ATPase activity was found to be reduced two-fold in presence of covalently closed circular duplex containing a mismatched base pair whereas, the activity was regained upon linearization of the circular duplex. This observation possibly suggests that the MutS clamps are trapped in the closed DNA heteroduplex. These studies, therefore, serve as the basis for a detailed investigation of the structure-function relationship among the protein partners of the mismatch repair pathway of H. influenzae.     

Structure and function of mismatch repair proteins

DNA mismatch repair is required for maintaining genomic stability and is highly conserved from prokaryotes to eukaryotes. Errors made during DNA replication, such as deletions, insertions and mismatched basepairs, are substrates for mismatch repair. Mismatch repair is strand-specific and targets only the newly synthesized daughter strand. To initiate mismatch repair in Escherichia coli, three proteins are essential, MutS, for mismatch recognition, MutH, for introduction of a nick in the target strand, and MutL, for mediating the interactions between MutH and MutS. Homologues of MutS and MutL important for mismatch repair have been found in nearly all organisms. Mutations in MutS and MutL homologues have been linked to increased cancer susceptibility in both mice and humans. Here, we review the crystal structures of the MutH endonuclease, a conserved ATPase fragment of MutL (LN40), and complexes of LN40 with various nucleotides. Based on the crystal structure, the active site of MutH has been identified and an evolutionary relationship between MutH and type II restriction endonucleases established. Recent crystallographic and biochemical studies have revealed that MutL operates as a molecular switch with its interactions with MutH and MutS regulated by ATP binding and hydrolysis. These crystal structures also shed light on the general mechanism of mismatch repair and the roles of Mut proteins in preventing mutagenesis.     

Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

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.     

Physical and functional interactions between Escherichia coli MutL and the Vsr repair endonuclease

DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways involved in the repair of T:G mismatches. To learn about competition and cooperation between these two repair pathways, we analyzed the physical and functional interaction between MutL and Vsr using biophysical and biochemical methods. Analytical ultracentrifugation reveals a nucleotide-dependent interaction between Vsr and the N-terminal domain of MutL. Using chemical crosslinking, we mapped the interaction site of MutL for Vsr to a region between the N-terminal domains similar to that described before for the interaction between MutL and the strand discrimination endonuclease MutH of the MMR system. Competition between MutH and Vsr for binding to MutL resulted in inhibition of the mismatch-provoked MutS- and MutL-dependent activation of MutH, which explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile MutS–MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or at the time of, activation of downstream effector molecules such as Vsr or MutH.     

The MutL ATPase is required for mismatch repair

Members of the MutL family contain a novel nucleotide binding motif near their amino terminus, and the Escherichia coli protein has been found to be a weak ATPase (Ban, C., and Yang, W. (1998) Cell 95, 541-552). Genetic analysis has indicated that substitution of Lys for Glu-32 within this motif of bacterial MutL results in a strong dominant negative phenotype (Aronshtam, A., and Marinus, M. G. (1996) Nucleic Acids Res. 24, 2498-2504). By in vitro comparison of MutL-E32K with the wild type protein, we show the mutant protein to be defective in DNA-activated ATP hydrolysis, as well as MutS- and MutL-dependent activation of the MutH d(GATC) endonuclease and the mismatch repair excision system. MutL-E32K also acts in dominant negative manner in the presence of wild type MutL in vitro, inhibiting the overall mismatch repair reaction, as well as MutH activation. As judged by protein affinity chromatography, MutL and MutL-E32K both support formation of ternary complexes that also contain MutS and MutH or MutS and DNA helicase II. These findings imply that the MutL nucleotide binding center is required for mismatch repair and suggest that the dominant negative behavior of the MutL-E32K mutation is due to the formation of dead-end complexes in which the MutL-E32K protein is unable to transduce a signal from MutS that otherwise results in mismatch-dependent activation of the MutH d(GATC) endonuclease or the unwinding activity of helicase II.     

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

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.     

The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair

The Escherichia coli MutS and MutL proteins have been conserved throughout evolution, although their combined functions in mismatch repair (MMR) are poorly understood. We have used biochemical and genetic studies to ascertain a physiologically relevant mechanism for MMR. The MutS protein functions as a regional lesion sensor. ADP-bound MutS specifically recognizes a mismatch. Repetitive rounds of mismatch-provoked ADP-->ATP exchange results in the loading of multiple MutS hydrolysis-independent sliding clamps onto the adjoining duplex DNA. MutL can only associate with ATP-bound MutS sliding clamps. Interaction of the MutS-MutL sliding clamp complex with MutH triggers ATP binding by MutL that enhances the endonuclease activity of MutH. Additionally, MutL promotes ATP binding-independent turnover of idle MutS sliding clamps. These results support a model of MMR that relies on two dynamic and redundant ATP-regulated molecular switches.     

The function of Asp70, Glu77 and Lys79 in the Escherichia coli MutH protein

The MutH protein, which is part of the Dam-directed mismatch repair system of Escherichia coli, introduces nicks in the unmethylated strand of a hemi-methylated DNA duplex. The latent endonuclease activity of MutH is activated by interaction with MutL, another member of the repair system. The crystal structure of MutH suggested that the active site residues include Asp70, Glu77 and Lys79, which are located at the bottom of a cleft where DNA binding probably occurs. We mutated these residues to alanines and found that the mutant proteins were unable to complement a chromosomal mutH deletion. The purified mutant proteins were able to bind to DNA with a hemi-methylated GATC sequence but had no detectable endonuclease activity with or without MutL. Although the data are consistent with the prediction of a catalytic role for Asp70, Glu77 and Lys79, it cannot be excluded that they are also involved in binding to MutL.     

Initiation of methyl-directed mismatch repair.

Escherichia coli MutH possesses an extremely weak d(GATC) endonuclease that responds to the state of methylation of the sequence (Welsh, K. M., Lu, A.-L., Clark, S., and Modrich, P. (1987) J. Biol. Chem. 262, 15624-15629). MutH endonuclease is activated in a reaction that requires MutS, MutL, ATP, and Mg2+ and depends upon the presence of a mismatch within the DNA. The degree of activation correlates with the efficiency with which a particular mismatch is subject to methyl-directed repair (G-T greater than G-G greater than A-C greater than C-C), and activated MutH responds to the state of DNA adenine methylation. Incision of an unmethylated strand occurs immediately 5' to a d(GATC) sequence, leaving 5' phosphate and 3' hydroxy termini (pN decreases pGpAp-TpC). Unmethylated d(GATC) sites are subject to double strand cleavage by activated MutH, an effect that may account for the killing of dam- mutants by 2-aminopurine. The mechanism of activation apparently requires ATP hydrolysis since adenosine-5'-O-(3-thiotriphosphate) not only fails to support the reaction but also inhibits activation promoted by ATP. The process has no obligate polarity as d(GATC) site incision by the activated nuclease can occur either 3' or 5' to the mismatch on an unmethylated strand. However, activation is sensitive to DNA topology. Circular heteroduplexes are better substrates than linear molecules, and activity of DNAs of the latter class depends on placement of the mismatch and d(GATC) site within the molecule. MutH activation is supported by a 6-kilobase linear heteroduplex in which the mismatch and d(GATC) site are centrally located and separated by 1 kilobase, but a related molecule, in which the two sites are located near opposite ends of the DNA, is essentially inactive as substrate. We conclude that MutH activation represents the initiation stage of methyl-directed repair and suggest that interaction of a mismatch and a d(GATC) site is provoked by MutS binding to a mispair, with subsequent ATP-dependent translocation of one or more Mut proteins along the helix leading to cleavage at a d(GATC) sequence on either side of the mismatch.     

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.     

Formation of a DNA mismatch repair complex mediated by ATP

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.     

The UvrD helicase and its modulation by the mismatch repair protein MutL

UvrD is a superfamily I DNA helicase with well documented roles in excision repair and methyl-directed mismatch repair (MMR) in addition to poorly understood roles in replication and recombination. The MutL protein is a homodimeric DNA-stimulated ATPase that plays a central role in MMR in Escherichia coli. This protein has been characterized as the master regulator of mismatch repair since it interacts with and modulates the activity of several other proteins involved in the mismatch repair pathway including MutS, MutH and UvrD. Here we present a brief summary of recent studies directed toward arriving at a better understanding of the interaction between MutL and UvrD, and the impact of this interaction on the activity of UvrD and its role in mismatch repair.     

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.     

In vitro and in vivo studies of MutS,MutL and MutH mutants: correlation of mismatch repair and DNA recombination

We have used the recently determined crystal structures of Escherichia coli (E. coli) MutS, MutL and MutH to guide construction of 47 amino-acid substitutions in these proteins and analyzed their behavior in mismatch repair and recombination in vitro and in vivo. We find that the active site of the MutH endonuclease is composed of regions from two separate structural domains and that the C-terminal 5 residues of MutH influence both DNA binding and cleavage. We also find that the non-specific DNA-binding activity of MutL is required for mismatch repair and probably functions after strand cleavage by MutH. Alteration of residues in either the mismatch recognition domain, the ATPase active site, or the domain interfaces linking the two activities can diminish the differential binding of MutS to homoduplex versus heteroduplex and results in the loss of mismatch-specific MutH activation. Finally, every mutation that abolishes mismatch repair is deficient in blocking homeologous recombination, suggesting that mismatch repair and prevention of homeologous recombination use the same MutS-MutL complexes for signaling in E. coli.     

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.     

DNA mismatch repair and mutation avoidance pathways

Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2-MSH6 (MutSalpha) and MSH2-MSH3 (MutSbeta). Interestingly, MSH3 (and thus MutSbeta) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1-PMS1 (MutLalpha) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases delta and epsilon. MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR.     

Evolution of the methyl directed mismatch repair system in Escherichia coli

DNA mismatch repair (MMR) repairs mispaired bases in DNA generated by replication errors. MutS or MutS homologs recognize mispairs and coordinate with MutL or MutL homologs to direct excision of the newly synthesized DNA strand. In most organisms, the signal that discriminates between the newly synthesized and template DNA strands has not been definitively identified. In contrast, Escherichia coli and some related gammaproteobacteria use a highly elaborated methyl-directed MMR system that recognizes Dam methyltransferase modification sites that are transiently unmethylated on the newly synthesized strand after DNA replication. Evolution of methyl-directed MMR is characterized by the acquisition of Dam and the MutH nuclease and by the loss of the MutL endonuclease activity. Methyl-directed MMR is present in a subset of Gammaproteobacteria belonging to the orders Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales, and a subset of the Alteromonadales (the EPVAA group) as well as in gammaproteobacteria that have obtained these genes by horizontal gene transfer, including the medically relevant bacteria Fluoribacter, Legionella, and Tatlockia and the marine bacteria Methylophaga and Nitrosococcus.     

Tyr212: a key residue involved in strand discrimination by the DNA mismatch repair endonuclease MutH

The molecular mechanism of how the dam-methylation status of the DNA is recognized during DNA mismatch repair by the strand discrimination endonuclease MutH is not known. A comparison of the crystal structure of MutH with those of co-crystal structures of several restriction endonucleases, together with a multiple sequence alignment of MutH and related proteins suggested that Phe94, Arg184 and Tyr212 could be involved in discrimination between a methylated or unmethylated adenine in the d(GATC) sequence. A mutational analysis revealed that the variants R184A and Y212S, but not F94A, were substantially reduced in their ability to complement a mismatch repair deficiency in a mutH(-) Escherichia coli strain. In vitro, R184A displayed a strongly reduced endonuclease activity, whereas the Y212S variant has almost completely lost its preference for cleaving the unmethylated strand at hemimethylated d(GATC) sites. Furthermore, the Y212 variant can cleave fully methlyated d(GATC) sites at a comparable rate to unmethylated d(GATC) sites. This demonstrates that Tyr212 is an important, if not the only amino acid residue in MutH for sensing the methylation status of the DNA.