MutL Activates UvrD by Interaction Between the MutL C-terminal Domain and the UvrD 2B Domain.

UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3'-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.

Identification of a Catalytic Lysine Residue Conserved Among GHKL ATPases: MutL, GyrB, and MORC.

DNA mismatch repair endonuclease MutL is a member of GHKL ATPase superfamily. Mutations of MutL homologs are causative of a hereditary cancer, Lynch syndrome. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although involvement of a basic residue had not been conceived in the catalytic mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is catalyzed by a residue with an alkaline pKa. Analyses of mutant aqMutLs showed that Lys79 is the catalytic residue, and the corresponding residues were confirmed to be critical for activities of human MutL homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations. Furthermore, it was confirmed that the catalytic lysine residue is conserved among DNA gyrases and microrchidia ATPases, other members of GHKL ATPases, indicating that the catalytic mechanism proposed here is applicable to these members of the superfamily.

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1.

MutL family proteins contain an N-terminal ATPase domain (NTD), an unstructured interdomain linker, and a C-terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand-specific DNA mismatch repair by cleaving the error-containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two- or three-metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C-terminal phenylalanine-glutamate-arginine-cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co-occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif-mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site.

Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation.

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

Regulation of the MLH1-MLH3 endonuclease in meiosis.

During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points9. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes4.

Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby.

DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS-MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS's frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL's ability to trap MutS, should increase the probability that MutS-MutL MMR initiation complexes localize near the mismatch.

A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL.

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69-1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

Dynamic human MutSα-MutLα complexes compact mismatched DNA.

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

Probing the DNA-binding center of the MutL protein from the Escherichia coli mismatch repair system via crosslinking and Förster resonance energy transfer.

As no crystal structure of full-size MutL bound to DNA has been obtained up to date, in the present work we used crosslinking and Förster resonance energy transfer (FRET) assays for probing the putative DNA-binding center of MutL from Escherichia coli. Several single-cysteine MutL variants (scMutL) were used for site-specific crosslinking or fluorophore modification. The crosslinking efficiency between scMutL proteins and mismatched DNA modified with thiol-reactive probes correlated with the distances from the Cys residues to the DNA calculated from a model of MutS-MutL-DNA complex. FRET-based investigation of DNA binding with different scMutL variants clearly showed that the highest signals were detected for the variants MutL(T218C) and MutL(A251C) indicating closeness of the positions 218 and 251 to DNA in the MutL-DNA complex. Indeed, the Cys218 and Cys251 of scMutL were crosslinked to the reactive DNA with the highest yield demonstrating their proximity to DNA in the MutL-DNA complex. The presence of MutS increased the yield of conjugate formation between the MutL variants and the modified DNA due to tighter MutL-DNA interactions caused by MutS binding to MutL.

Human MutLγ, the MLH1-MLH3 heterodimer, is an endonuclease that promotes DNA expansion.

MutL proteins are ubiquitous and play important roles in DNA metabolism. MutLγ (MLH1-MLH3 heterodimer) is a poorly understood member of the eukaryotic family of MutL proteins that has been implicated in triplet repeat expansion, but its action in this deleterious process has remained unknown. In humans, triplet repeat expansion is the molecular basis for ∼40 neurological disorders. In addition to MutLγ, triplet repeat expansion involves the mismatch recognition factor MutSß (MSH2-MSH3 heterodimer). We show here that human MutLγ is an endonuclease that nicks DNA. Strikingly, incision of covalently closed, relaxed loop-containing DNA by human MutLγ is promoted by MutSß and targeted to the strand opposite the loop. The resulting strand break licenses downstream events that lead to a DNA expansion event in human cell extracts. Our data imply that the mammalian MutLγ is a unique endonuclease that can initiate triplet repeat DNA expansions.

UvrD helicase activation by MutL involves rotation of its 2B subdomain.

Escherichia coli UvrD is a superfamily 1 helicase/translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an Escherichia coli MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer-DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain.

Binding of the regulatory domain of MutL to the sliding ß-clamp is species specific.

The ß-clamp is a protein hub central to DNA replication and fork management. Proteins interacting with the ß-clamp harbor a conserved clamp-binding motif that is often found in extended regions. Therefore, clamp interactions have -almost exclusively- been studied using short peptides recapitulating the binding motif. This approach has revealed the molecular determinants that mediate the binding but cannot describe how proteins with clamp-binding motifs embedded in structured domains are recognized. The mismatch repair protein MutL has an internal clamp-binding motif, but its interaction with the ß-clamp has different roles depending on the organism. In Bacillus subtilis, the interaction stimulates the endonuclease activity of MutL and it is critical for DNA mismatch repair. Conversely, disrupting the interaction between Escherichia coli MutL and the ß-clamp only causes a mild mutator phenotype. Here, we determined the structures of the regulatory domains of E. coli and B. subtilis MutL bound to their respective ß-clamps. The structures reveal different binding modes consistent with the binding to the ß-clamp being a two-step process. Functional characterization indicates that, within the regulatory domain, only the clamp binding motif is required for the interaction between the two proteins. However, additional motifs beyond the regulatory domain may stabilize the interaction. We propose a model for the activation of the endonuclease activity of MutL in organisms lacking methyl-directed mismatch repair.

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.

The endonuclease domain of Bacillus subtilis MutL is functionally asymmetric.

DNA mismatch repair is an evolutionarily conserved repair pathway that corrects replication errors. In most prokaryotes and all eukaryotes, the mismatch repair protein MutL is a sequence-unspecific endonuclease that nicks the newly synthesized strand and marks it for repair. Although the sequence of the endonuclease domain of MutL is not conserved, eukaryotic MutLα and prokaryotic MutL share four conserved motifs that define the endonuclease site of the protein. Their endonuclease activity is stimulated by the processivity sliding ß-clamp, or its eukaryotic counterpart PCNA, highlighting the functional conservation. Bacterial MutL homologs form homodimers and, therefore, they have two endonuclease sites. However, eukaryotic MutL homologs associate to form heterodimers, where only one of the protomers of the dimer has endonuclease activity. To probe whether bacterial MutL needs its two endonuclease sites, we engineered variants of B. subtilis MutL harboring a single nuclease site and showed that these variants are functional nucleases. We also find that the protomer harboring the nuclease site must be able to bind to the ß-clamp to recapitulate the nicking activity of wild-type MutL. These results demonstrate the functional asymmetry of bacterial MutL and strengthen the similarities with the endonuclease activity of eukaryotic MutL homologs.

DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477).

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.

The mutagen and carcinogen cadmium is a high-affinity inhibitor of the zinc-dependent MutLα endonuclease.

MutLα (MLH1-PMS2 heterodimer), which acts as a strand-directed endonuclease during the initiation of eukaryotic mismatch repair, has been postulated to function as a zinc-dependent enzyme [Kosinski J, Plotz G, Guarné A, Bujnicki JM, Friedhoff P (2008) J Mol Biol 382:610-627]. We show that human MutLα copurifies with two bound zinc ions, at least one of which resides within the endonuclease active site, and that bound zinc is required for endonuclease function. Mutagenic action of the carcinogen cadmium, a known inhibitor of zinc-dependent enzymes, is largely due to selective inhibition of mismatch repair [Jin YH, et al. (2003) Nat Genet 34:326-329]. We show that cadmium is a potent inhibitor (apparent Ki ∼ 200 nM) of MutLα endonuclease and that cadmium inhibition is reversed by zinc. We also show that inhibition of mismatch repair in cadmium-treated nuclear extract is significantly reversed by exogenous MutLα but not by MutSα (MSH2-MSH6 heterodimer) and that MutLα reversal depends on integrity of the endonuclease active site. Exogenous MutLα also partially rescues the mismatch repair defect in nuclear extract prepared from cells exposed to cadmium. These findings indicate that targeted inhibition of MutLα endonuclease contributes to cadmium inhibition of mismatch repair. This effect may play a role in the mechanism of cadmium carcinogenesis.

GAA•TTC repeat expansion in human cells is mediated by mismatch repair complex MutLγ and depends upon the endonuclease domain in MLH3 isoform one.

DNA repeat expansion underlies dozens of progressive neurodegenerative disorders. While the mechanisms driving repeat expansion are not fully understood, increasing evidence suggests a central role for DNA mismatch repair. The mismatch repair recognition complex MutSß (MSH2-MSH3) that binds mismatched bases and/or insertion/deletion loops has previously been implicated in GAA•TTC, CAG•CTG and CGG•CCG repeat expansion, suggesting a shared mechanism. MutSß has been studied in a number of models, but the contribution of subsequent steps mediated by the MutL endonuclease in this pathway is less clear. Here we show that MutLγ (MLH1-MLH3) is the MutL complex responsible for GAA•TTC repeat expansion. Lentiviral expression of shRNA targeting MutL nuclease components MLH1, PMS2, and MLH3 revealed that reduced expression of MLH1 or MLH3 reduced the repeat expansion rate in a human Friedreich ataxia cell model, while targeting PMS2 did not. Using splice-switching oligonucleotides we show that MLH3 isoform 1 is active in GAA•TTC repeat expansion while the nuclease-deficient MLH3 isoform 2 is not. MLH3 isoform switching slowed repeat expansion in both model cells and FRDA patient fibroblasts. Our work indicates a specific and active role for MutLγ in the expansion process and reveals plausible targets for disease-modifying therapies.

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair.

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes.

Crystal structure and DNA-binding property of the ATPase domain of bacterial mismatch repair endonuclease MutL from Aquifex aeolicus.

DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-ß sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule.

Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutLγ complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.