Differential ATP binding and intrinsic ATP hydrolysis by amino-terminal domains of the yeast Mlh1 and Pms1 proteins.

MutL homologs belong to a family of proteins that share a conserved ATP binding site. We demonstrate that amino-terminal domains of the yeast MutL homologs Mlh1 and Pms1 required for DNA mismatch repair both possess independent, intrinsic ATPase activities. Amino acid substitutions in the conserved ATP binding sites concomitantly reduce ATP binding, ATP hydrolysis, and DNA mismatch repair in vivo. The ATPase activities are weak, consistent with the hypothesis that ATP binding is primarily responsible for modulating interactions with other MMR components. Three approaches, ATP hydrolysis assays, limited proteolysis protection, and equilibrium dialysis, provide evidence that the amino-terminal domain of Mlh1 binds ATP with >10-fold higher affinity than does the amino-terminal domain of Pms1. This is consistent with a model wherein ATP may first bind to Mlh1, resulting in events that permit ATP binding to Pms1 and later steps in DNA 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.     

Functional domains of the Saccharomyces cerevisiae Mlh1p and Pms1p DNA mismatch repair proteins and their relevance to human hereditary nonpolyposis colorectal cancer-associated mutations.

The MutL protein is an essential component of the Escherichia coli methyl-directed mismatch repair system but has no known enzymatic function. In the yeast Saccharomyces cerevisiae, the MutL equivalent, an Mlh1p and Pms1p heterodimer, interacts with Msh2p bound to mismatch-containing DNA. Little is known of the functional domains of Mlh1p and Pms1p. In this report, we define the Mlh1p and Pms1p domains required for Mlh1p-Pms1p interaction. The Mlh1p-interactive domain of Pms1p is comprised of 260 amino acids near the carboxyl terminus while the Pms1p-interactive domain of Mlh1p resides in the final 212 residues. The two domains are sufficient for Mlh1p-Pms1p interaction, as determined by the two-hybrid assay and by in vitro protein affinity chromatography. Deletions within the domains completely eliminated Mlh1p-Pms1p interaction. Using site-directed mutagenesis, we altered a number of highly conserved residues in the Mlh1p and Pms1p proteins, including some alterations that mimic germline mutations observed for human hereditary nonpolyposis colorectal cancer. Alterations either in the consensus MutL box located in the amino-terminal portion of each protein or in the carboxyl-terminal homology motif of Mlh1p eliminated DNA mismatch repair function but had no effect on Mlh1p-Pms1p interaction. In addition, certain MLH1 and PMS1 mutant alleles caused a dominant negative mutator effect when overexpressed. We discuss the implications of these findings for the structural organization of the Mlh1p and Pms1p proteins and the importance of Mlh1p-Pms1p interaction.     

Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha

Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLalpha, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH(2) termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLalpha, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLalpha demonstrated that the NH(2) terminus of MutLalpha undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH(2) termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLalpha undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.     

Contribution of human mlh1 and pms2 ATPase activities to DNA mismatch repair

MutLalpha, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLalpha and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLalpha proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLalpha demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLalpha activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.     

The Unstructured Linker Arms of Mlh1-Pms1 Are Important for Interactions with DNA during Mismatch Repair

DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) proteins (primarily Mlh1-Pms1 in baker's yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20-30nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker's yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR.     

Endonuclease activities of MutLα and its homologs in DNA mismatch repair

MutLα is a key component of the DNA mismatch repair system in eukaryotes. The DNA mismatch repair system has several genetic stabilization functions. Of these functions, DNA mismatch repair is the major one. The loss of MutLα abolishes DNA mismatch repair, thereby predisposing humans to cancer. MutLα has an endonuclease activity that is required for DNA mismatch repair. The endonuclease activity of MutLα depends on the DQHA(X)₂E(X)₄E motif which is a part of the active site of the nuclease. This motif is also present in many bacterial MutL and eukaryotic MutLγ proteins, DNA mismatch repair system factors that are homologous to MutLα. Recent studies have shown that yeast MutLγ and several MutL proteins containing the DQHA(X)₂E(X)₄E motif possess endonuclease activities. Here, we review the endonuclease activities of MutLα and its homologs in the context of DNA mismatch repair.     

Supercomplex formation between Mlh1-Mlh3 and Sgs1-Top3 heterocomplexes in meiotic yeast cells

The genome of Saccharomyces cerevisia encodes four mismatch repair MutL proteins and these proteins form three heterocomplexes: Mlh1-Mlh2, Mlh1-Mlh3, and Mlh1-Pms1. Only, the Mlh1-Mlh3 heterocomplex has been implicated specifically in promotion of meiotic crossing-over. In this report, we show that yeast Mlh3 co-immunoprecipitates with Sgs1 helicase in sporulating cells at late stage of meiotic prophase I. Sgs1, a member of the RecQ DNA helicase family, appears to form a stable complex with topoisomerase III (Top3) during meiosis. We suggest that Mlh1-Mlh3 heterocomplex may act as a molecular matchmaker to coordinate Sgs1-Top3 complex in the resolution of meiotic recombination intermediates.     

Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD.

UvrD (DNA helicase II) is an essential component of two major DNA repair pathways in Escherichia coli: methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. In addition, it has an undefined role in the RecF recombination pathway and possibly in replication. In an effort to better understand the role of UvrD in these various aspects of DNA metabolism, a yeast two-hybrid screen was used to search for interacting protein partners. Screening of an E.coli genomic library revealed a potential interaction between UvrD and MutL, a component of the methyl-directed mismatch repair pathway. The interaction was confirmed by affinity chromatography using purified proteins. Deletion analysis demonstrated that the C-terminal 218 amino acids (residues 398-615) of MutL were sufficient to produce the two-hybrid interaction with UvrD. On the other hand, both the N- and C-termini of UvrD were required for interaction with MutL. The implications of this interaction for the mismatch repair mechanism are discussed.     

Single-molecule analysis reveals the kinetics and physiological relevance of MutL-ssDNA binding

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (K_D = 29 nM) while it dramatically decreases above 100 mM (K_D>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.     

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.     

Functional residues on the surface of the N-terminal domain of yeast Pms1

Saccharomyces cerevisiae MutLα is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5 Å crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.     

Alleles of the yeast Pms1 mismatch-repair gene that differentially affect recombination- and replication-related processes

Mismatch-repair (MMR) systems promote eukaryotic genome stability by removing errors introduced during DNA replication and by inhibiting recombination between nonidentical sequences (spellchecker and antirecombination activities, respectively). Following a common mismatch-recognition step effected by MutS-homologous Msh proteins, homologs of the bacterial MutL ATPase (predominantly the Mlh1p-Pms1p heterodimer in yeast) couple mismatch recognition to the appropriate downstream processing steps. To examine whether the processing steps in the spellchecker and antirecombination pathways might differ, we mutagenized the yeast PMS1 gene and screened for mitotic separation-of-function alleles. Two alleles affecting only the antirecombination function of Pms1p were identified, one of which changed an amino acid within the highly conserved ATPase domain. To more specifically address the role of ATP binding/hydrolysis in MMR-related processes, we examined mutations known to compromise the ATPase activity of Pms1p or Mlh1p with respect to the mitotic spellchecker and antirecombination activities and with respect to the repair of mismatches present in meiotic recombination intermediates. The results of these analyses confirm a differential requirement for the Pms1p ATPase activity in replication vs. recombination processes, while demonstrating that the Mlh1p ATPase activity is important for all examined MMR-related functions.     

Systematic mutagenesis of the Saccharomyces cerevisiae MLH1 gene reveals distinct roles for Mlh1p in meiotic crossing over and in vegetative and meiotic mismatch repair

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.     

Saccharomyces cerevisiae MutLalpha is a mismatch repair endonuclease

MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLalpha (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLalpha, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLalpha is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSalpha, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLalpha endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLalpha ATPase activity or the ability of the protein to support assembly of the yMutLalpha.yMutSalpha.heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLalpha incision to the discontinuous strand of a nicked heteroduplex.     

Homologs of MutS and MutL during mammalian meiosis

In eukaryotes, homologs of the Escherichia coli MutS and MutL proteins are crucial for both meiotic recombination and post-replicative DNA mismatch repair. Both pathways require the formation of a MutS homolog complex which interacts with a second heterodimer, composed of two MutL homologs. During mammalian meiosis, it is likely that chromosome synapsis requires the presence of a MSH4-MSH5 heterodimer. PMS2, a MutL homolog, seems to play an important role in this process. A MSH4-MSH5 heterodimer is also likely present later with other MutL homologs (MLH1 and MLH3) and is involved in the crossing-over process. The phenotype of msh4-/- mutant mice and MSH4 immunolocalization on meiotic chromosomes suggest that MSH4 has an early function in mammalian meiotic recombination. Both MSH4 and PMS2 directly interact with the RAD51 DNA strand exchange protein. In addition, MSH4 and RAD51 proteins co-localize on mouse meiotic chromosome cores. These results suggest that MSH4 and its partners could act, just after strand exchange promoted by RAD51, to check the homology of DNA heteroduplexes.     

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.     

Regulation of mitotic homeologous recombination in yeast. Functions of mismatch repair and nucleotide excision repair genes

The Saccharomyces cerevisiae homologs of the bacterial mismatch repair proteins MutS and MutL correct replication errors and prevent recombination between homeologous (nonidentical) sequences. Previously, we demonstrated that Msh2p, Msh3p, and Pms1p regulate recombination between 91% identical inverted repeats, and here use the same substrates to show that Mlh1p and Msh6p have important antirecombination roles. In addition, substrates containing defined types of mismatches (base-base mismatches; 1-, 4-, or 12-nt insertion/deletion loops; or 18-nt palindromes) were used to examine recognition of these mismatches in mitotic recombination intermediates. Msh2p was required for recognition of all types of mismatches, whereas Msh6p recognized only base-base mismatches and 1-nt insertion/deletion loops. Msh3p was involved in recognition of the palindrome and all loops, but also had an unexpected antirecombination role when the potential heteroduplex contained only base-base mismatches. In contrast to their similar antimutator roles, Pms1p consistently inhibited recombination to a lesser degree than did Msh2p. In addition to the yeast MutS and MutL homologs, the exonuclease Exo1p and the nucleotide excision repair proteins Rad1p and Rad10p were found to have roles in inhibiting recombination between mismatched substrates.     

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.