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.     

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.     

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.     

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.     

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     

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.     

The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA.

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.     

The beta sliding clamp binds to multiple sites within MutL and MutS

The MutL and MutS proteins are the central components of the DNA repair machinery that corrects mismatches generated by DNA polymerases during synthesis. We find that MutL interacts directly with the beta sliding clamp, a ring-shaped dimeric protein that confers processivity to DNA polymerases by tethering them to their substrates. Interestingly, the interaction of MutL with beta only occurs in the presence of single-stranded DNA. We find that the interaction occurs via a loop in MutL near the ATP-binding site. The binding site of MutL on beta locates to the hydrophobic pocket between domains two and three of the clamp. Site-specific replacement of two residues in MutL diminished interaction with beta without disrupting MutL function with helicase II. In vivo studies reveal that this mutant MutL is no longer functional in mismatch repair. In addition, the human MLH1 has a close match to the proliferating cell nuclear antigen clamp binding motif in the region that corresponds to the beta interaction site in Escherichia coli MutL, and a peptide corresponding to this site binds proliferating cell nuclear antigen. The current report also examines in detail the interaction of beta with MutS. We find that two distinct regions of MutS interact with beta. One is located near the C terminus and the other is close to the N terminus, within the mismatch binding domain. Complementation studies using genes encoding different MutS mutants reveal that the N-terminal beta interaction motif on MutS is essential for activity in vivo, but the C-terminal interaction site for beta is not. In light of these results, we propose roles for the beta clamp in orchestrating the sequence of events that lead to mismatch repair in the cell.     

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 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.     

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.     

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.     

DnaN clamp zones provide a platform for spatiotemporal coupling of mismatch detection to DNA replication

Mismatch repair (MMR) increases the fidelity of DNA replication by identifying and correcting replication errors. Processivity clamps are vital components of DNA replication and MMR, yet the mechanism and extent to which they participate in MMR remains unclear. We investigated the role of the Bacillus subtilis processivity clamp DnaN, and found that it serves as a platform for mismatch detection and coupling of repair to DNA replication. By visualizing functional MutS fluorescent fusions in vivo, we find that MutS forms foci independent of mismatch detection at sites of replication (i.e. the replisome). These MutS foci are directed to the replisome by DnaN clamp zones that aid mismatch detection by targeting the search to nascent DNA. Following mismatch detection, MutS disengages from the replisome, facilitating repair. We tested the functional importance of DnaN‐mediated mismatch detection for MMR, and found that it accounts for 90% of repair. This high dependence on DnaN can be bypassed by increasing MutS concentration within the cell, indicating a secondary mode of detection in vivo whereby MutS directly finds mismatches without associating with the replisome. Overall, our results provide new insight into the mechanism by which DnaN couples mismatch recognition to DNA replication in living cells.     

Residues in the N-Terminal Domain of MutL Required for Mismatch Repair in Bacillus subtilis

Mismatch repair is a highly conserved pathway responsible for correcting DNA polymerase errors incorporated during genome replication. MutL is a mismatch repair protein known to coordinate several steps in repair that ultimately results in strand removal following mismatch identification by MutS. MutL homologs from bacteria to humans contain well-conserved N-terminal and C-terminal domains. To understand the contribution of the MutL N-terminal domain to mismatch repair, we analyzed 14 different missense mutations in Bacillus subtilis MutL that were conserved with missense mutations identified in the human MutL homolog MLH1 from patients with hereditary nonpolyposis colorectal cancer (HNPCC). We characterized missense mutations in or near motifs important for ATP binding, ATPase activity, and DNA binding. We found that 13 of the 14 missense mutations conferred a substantial defect to mismatch repair in vivo, while three mutant alleles showed a dominant negative increase in mutation frequency to wild-type mutL. We performed immunoblot analysis to determine the relative stability of each mutant protein in vivo and found that, although most accumulated, several mutant proteins failed to maintain wild-type levels, suggesting defects in protein stability. The remaining missense mutations located in areas of the protein important for DNA binding, ATP binding, and ATPase activities of MutL compromised repair in vivo. Our results define functional residues in the N-terminal domain of B. subtilis MutL that are critical for mismatch repair in vivo.     

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.     

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.     

Discrete in vivo roles for the MutL homologs Mlh2p and Mlh3p in the removal of frameshift intermediates in budding yeast

The DNA mismatch repair machinery is involved in the correction of a wide variety of mutational intermediates. In bacterial cells, homodimers of the MutS protein bind mismatches and MutL homodimers couple mismatch recognition to downstream processing steps [1]. Eukaryotes possess multiple MutS and MutL homologs that form discrete, heterodimeric complexes with specific mismatch recognition and repair properties. In yeast, there are six MutS (Msh1-6p) and four MutL (Mlh1-3p and Pms1p) family members [2] [3]. Heterodimers comprising Msh2p and Msh3p or Msh2p and Msh6p recognize mismatches in nuclear DNA [4] [5] and the subsequent processing steps most often involve a Mlh1p-Pms1P heterodimer [6] [7]. Mlh1p also forms heterodimeric complexes with Mlh2p and Mlh3p [8], and a minor role for Mlh3p in nuclear mismatch repair has been reported [9]. No mismatch repair function has yet been assigned to the fourth yeast MutL homolog, Mlh2p, although mlh2 mutants exhibit weak resistance to some DNA damaging agents [10]. We have used two frameshift reversion assays to examine the roles of the yeast Mlh2 and Mlh3 proteins in vivo. This analysis demonstrates, for the first time, that yeast Mlh2p plays a role in the repair of mutational intermediates, and extends earlier results implicating Mlh3p in mismatch repair.     

ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL.

Functional interactions of Escherichia coli MutS and MutL in mismatch repair are dependent on ATP. In this study, we show that MutS and MutL associate with immobilised DNA in a manner dependent on ATP hydrolysis and with an ATP concentration near the solution K m of the ATPase of MutS. After removal of MutS, MutL and ATP, much of the protein in this ternary complex is not stably associated, with MutL leaving the complex more rapidly than MutS. The rapid dissociation reveals a dynamic interaction with concurrent rapid association and dissociation of proteins from the DNA. Analysis by surface plasmon resonance showed that the DNA interacting with dynamically bound protein was more resistant to nuclease digestion than the DNA in MutS-DNA complexes. Non-hydrolysable analogs of ATP inhibit the formation of this dynamic complex, but permit formation of a second type of ternary complex with MutS and MutL stably bound to the immobilised DNA.     

MutS mediates heteroduplex loop formation by a translocation mechanism.

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.     

Chemical trapping of the dynamic MutS-MutL complex formed in DNA mismatch repair in Escherichia coli

The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain.