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.     

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.     

Distinct MutS DNA-binding modes that are differentially modulated by ATP binding and hydrolysis.

The role of MutS ATPase in mismatch repair is controversial. To clarify further the function of this activity, we have examined adenine nucleotide effects on interactions of Escherichia coli MutS with homoduplex and heteroduplex DNAs. In contrast to previous results with human MutS alpha, we find that a physical block at one end of a linear heteroduplex is sufficient to support stable MutS complex formation in the presence of ATP.Mg(2+). Surface plasmon resonance analysis at low ionic strength indicates that the lifetime of MutS complexes with heteroduplex DNA depends on the nature of the nucleotide present when MutS binds. Whereas complexes prepared in the absence of nucleotide or in the presence of ADP undergo rapid dissociation upon challenge with ATP x Mg(2+), complexes produced in the presence of ATP x Mg(2+), adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) x Mg(2+), or ATP (no Mg(2+)) are resistant to dissociation upon ATP challenge. AMPPNP x Mg(2+) and ATP (no Mg(2+)) reduce MutS affinity for heteroduplex but have little effect on homoduplex affinity, resulting in abolition of specificity for mispaired DNA at physiological salt concentrations. Conversely, the highest mismatch specificity is observed in the absence of nucleotide or in the presence of ADP. ADP has only a limited effect on heteroduplex affinity but reduces MutS affinity for homoduplex DNA.     

Electrochemical detection of mismatched DNA using a MutS probe

A direct and label-free electrochemical biosensor for the detection of the protein–mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GT > CT > CC ≈ AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.     

Modulation of MutS ATP hydrolysis by DNA cofactors

Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.     

Mechanism for verification of mismatched and homoduplex DNAs by nucleotides‐bound MutS analyzed by molecular dynamics simulations

In order to understand how MutS recognizes mismatched DNA and induces the reaction of DNA repair using ATP, the dynamics of the complexes of MutS (bound to the ADP and ATP nucleotides, or not) and DNA (with mismatched and matched base‐pairs) were investigated using molecular dynamics simulations. As for DNA, the structure of the base‐pairs of the homoduplex DNA which interacted with the DNA recognition site of MutS was intermittently disturbed, indicating that the homoduplex DNA was unstable. As for MutS, the disordered loops in the ATPase domains, which are considered to be necessary for the induction of DNA repair, were close to (away from) the nucleotide‐binding sites in the ATPase domains when the nucleotides were (not) bound to MutS. This indicates that the ATPase domains changed their structural stability upon ATP binding using the disordered loop. Conformational analysis by principal component analysis showed that the nucleotide binding changed modes which have structurally solid ATPase domains and the large bending motion of the DNA from higher to lower frequencies. In the MutS–mismatched DNA complex bound to two nucleotides, the bending motion of the DNA at low frequency modes may play a role in triggering the formation of the sliding clamp for the following DNA‐repair reaction step. Moreover, MM‐PBSA/GBSA showed that the MutS–homoduplex DNA complex bound to two nucleotides was unstable because of the unfavorable interactions between MutS and DNA. This would trigger the ATP hydrolysis or separation of MutS and DNA to continue searching for mismatch base‐pairs. Proteins 2016; 84:1287–1303. © 2016 Wiley Periodicals, Inc.     

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.     

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.     

A single mismatch in the DNA induces enhanced aggregation of MutS. Hydrodynamic analyses of the protein-DNA complexes

Changes in the oligomeric status of MutS protein was probed in solution by dynamic light scattering (DLS), and corroborated by sedimentation analyses. In the absence of any nucleotide cofactor, free MutS protein [hydrodynamic radius (Rh) of 10-12 nm] shows a small increment in size (Rh 14 nm) following the addition of homoduplex DNA (121 bp), whereas the same increases to about 18-20 nm with heteroduplex DNA containing a mismatch. MutS forms large aggregates (Rh > 500 nm) with ATP, but not in the presence of a poorly hydrolysable analogue of ATP (ATPgammaS). Addition of either homo- or heteroduplex DNA attenuates the same, due to protein recruitment to DNA. However, the same protein/DNA complexes, at high concentration of ATP (10 mm), manifest an interesting property where the presence of a single mismatch provokes a much larger oligomerization of MutS on DNA (Rh > 500 nm in the presence of MutL) as compared to the normal homoduplex (Rh approximately 100-200 nm) and such mismatch induced MutS aggregation is entirely sustained by the ongoing hydrolysis of ATP in the reaction. We speculate that the surprising property of a single mismatch, in nucleating a massive aggregation of MutS encompassing the bound DNA might play an important role in mismatch repair system.     

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.     

Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism<Emphasis Type="Italic"> Deinococcus radiodurans</Emphasis>

We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous Rif^R mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration—suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to -rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Baldacci     

Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutSalpha is abolished by a single mutation in the hMSH6 subunit

The most abundant mismatch binding factor in human cells, hMutSalpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein. The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5)DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A., Biswas, I., Camerini-Otero, R. D., and Hsieh, P. (1997) J. Biol. Chem. 272, 23811-23817). We introduced an identical mutation into one or both subunits of hMutSalpha. The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutSalpha were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA. Thus the N-terminal portion of hMSH6 appears to impart on hMutSalpha not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA.     

Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutS gene, implicated in DNA mismatch repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa. Its predicted amino acid sequence showed 56 and 39% homology with Escherichia coli MutS and human hMsh2 proteins, respectively. The T.thermophilus mutS gene complemented the hypermutability of the E.coli mutS mutant, suggesting that T.thermophilus MutS protein was active in E.coli and could interact with E.coli MutL and/or MutH proteins. The T.thermophilus mutS gene product was overproduced in E.coli and then purified to homogeneity. Its molecular mass was estimated to be 91 kDa by SDS-PAGE but approx. 330 kDa by size-exclusion chromatography, suggesting that T.thermophilus MutS protein was a tetramer in its native state. Circular dichroic measurements indicated that this protein had an alpha-helical content of approx. 50%, and that it was stable between pH 1.5 and 12 at 25 degree C and was stable up to 80 degree C at neutral pH. Thermus thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80 degrees C. The kinetic parameters of the ATPase activity at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(-1). Thermus thermophilus MutS protein bound specifically with G-T mismatched DNA even at 60 degrees C.     

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.     

Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA

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; S1(ADP)-S2(ATP) is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1(ATP)-S2(ATP) and S1(ATP)-S2(ADP) 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.     

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.     

ATP increases the affinity between MutS ATPase domains. Implications for ATP hydrolysis and conformational changes

MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.     

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.     

Trapping and visualizing intermediate steps in the mismatch repair pathway in vivo

During mismatch repair, MutS is responsible for mismatch detection and the recruitment of MutL to the mismatch through a mechanism that is unknown in most organisms. Here, we identified a discrete site on MutS that is occupied by MutL in Bacillus subtilis. The MutL binding site is composed of two adjacent phenylalanine residues located laterally in an exposed loop of MutS. Disruption of this site renders MutS defective in binding MutL in vitro and in vivo, while also eliminating mismatch repair. Analysis of MutS repair complexes in vivo shows that MutS mutants defective in interaction with MutL are ‘trapped’ in a repetitive loading response. Furthermore, these mutant MutS repair complexes persist on DNA away from the DNA polymerase, suggesting that MutS remains loaded on mismatch proximal DNA awaiting arrival of MutL. We also provide evidence that MutS and MutL interact independent of mismatch binding by MutS in vivo and in vitro, suggesting that MutL can transiently probe MutS to determine if MutS is mismatch bound. Together, these data provide insights into the mechanism that MutS employs to recruit MutL, and the consequences that ensue when MutL recruitment is blocked.     

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.