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 endonuclease domain of MutL interacts with the β sliding clamp

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 Bacillus subtilis and Escherichia 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.     

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.     

Deletion mutation analysis of the mutS gene in Escherichia coli

The MutS protein is part of the dam-directed MutHLS mismatch repair pathway in Escherichia coli. We have constructed deletion derivatives in the mutS gene, which retain the P-loop coding region for ATP binding. The mutant proteins were assayed for ATP hydrolysis, heteroduplex DNA binding, heterodimer MutS formation, and the ability to interact with MutL. Dimerization was assayed by expressing His6-tagged wild-type and non-tagged deletion mutant proteins in the same cell and isolating the His6-tagged protein followed by MutS immunoblotting after SDS-polyacrylamide gel electrophoresis. MutS-MutL interaction was measured using the same technique except that the MutL protein carried the His6 tag. Our results indicate that DNA binding ability resides in the N-terminal end of MutS, and dimerization and MutL interactions are located in the C-terminal end. Given the extensive amino acid homology in the MutS family our results with E. coli should be applicable to MutS homologues in other prokaryotes and eukaryotes.     

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.     

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.     

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.     

Visualization of mismatch repair in bacterial cells.

We determined the localizations of mismatch repair proteins in living Bacillus subtilis cells. MutS-GFP colocalized with the chromosome in all cells and formed foci in a subset of cells. MutL-GFP formed foci in a subset of cells, and its localization was MutS dependent. The introduction of mismatches by growth in 2-aminopurine caused a replication-dependent increase in the number of cells with MutS and MutL foci. Approximately half of the MutS foci colocalized with DNA polymerase foci. We conclude that MutS is associated with the entire chromosome, poised to detect mismatches. After detection, it appears that mismatch repair foci assemble at mismatches as they emerge from the DNA polymerase and are then carried away from the replisome by continuing replication.     

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.     

MutL-catalyzed ATP hydrolysis is required at a post-UvrD loading step in methyl-directed mismatch repair

Methyl-directed mismatch repair is a coordinated process that ensures replication fidelity and genome integrity by resolving base pair mismatches and insertion/deletion loops. This post-replicative event involves the activities of several proteins, many of which appear to be regulated by MutL. MutL interacts with and modulates the activities of MutS, MutH, UvrD, and perhaps other proteins. The purified protein catalyzes a slow ATP hydrolysis reaction that is essential for its role in mismatch repair. However, the role of the ATP hydrolysis reaction is not understood. We have begun to address this issue using two point mutants: MutL-E29A, which binds nucleotide but does not catalyze ATP hydrolysis, and MutL-D58A, which does not bind nucleotide. As expected, both mutants failed to complement the loss of MutL in genetic assays. Purified MutL-E29A protein interacted with MutS and stimulated the MutH-catalyzed nicking reaction in a mismatch-dependent manner. Importantly, MutL-E29A stimulated the loading of UvrD on model substrates. In fact, stimulation of UvrD-catalyzed unwinding was more robust with MutL-E29A than the wild-type protein. MutL-D58A, on the other hand, did not interact with MutS, stimulate MutH-catalyzed nicking, or stimulate the loading of UvrD. We conclude that ATP-bound MutL is required for the incision steps associated with mismatch repair and that ATP hydrolysis by MutL is required for a step in the mismatch repair pathway subsequent to the loading of UvrD and may serve to regulate helicase loading.     

Some Features of Base Pair Mismatch and Heterology Repair in Escherichia coli

We have used artificially constructed heteroallelic heteroduplex molecules of bacteriophage lambda DNA to transfect Escherichia coli, and E. coli mutants deficient in various functions involved in the adenine methylation-directed mismatch repair system, MutL, MutS, MutH, and UvrD (MutU). Analysis of the allele content of single infective centers shows that this repair system often acts on several mismatches, separated by as many as 2000 bp, on one of the strands of a heteroduplex molecule. When the methyl-directed mismatch repair system is disabled by mutH or uvrD mutations, localized mismatch repair becomes prominent. This prominent localized repair that can result in separation of very closely linked markers requires the functions MutL and MutS, is independent of adenine methylation, and appears to reflect another mechanism of mismatch repair. Heterology-containing heteroduplex molecules with a deletion in one strand often escape processing. However, when the heterology includes the stem and loop structure of a transposon, Tn10, the transposon is lost.     

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.     

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.     

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS–MutL complex recruits the Uvr proteins to complete repair.     

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.     

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.     

Dominant negative mutator mutations in the mutL gene of Escherichia coli.

The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.     

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.     

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.