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.     

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.     

Reconstitution of 5'-directed human mismatch repair in a purified system

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.     

Dimerization of MLH1 and PMS2 limits nuclear localization of MutLalpha

DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails. The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation. Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLalpha (the MLH1-PMS2 dimer). We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLalpha. Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS. Limited nuclear localization of MutLalpha may thus represent a novel mechanism by which cells fine-tune mismatch repair functions. This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.     

Human MutLalpha: the jack of all trades in MMR is also an endonuclease

Recently, Paul Modrich's group reported the discovery of an intrinsic endonuclease activity for human MutLalpha. This breakthrough provides a satisfactory answer to the longstanding puzzle of a missing nuclease activity in human mismatch repair and will undoubtedly lead to new investigations of DNA repair and replication. Here, the implications of this exciting new finding are discussed in the context of mismatch repair in Escherichia coli and humans.     

Endonucleolytic function of MutLalpha in human mismatch repair

Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5'-to-3' activity of MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.     

Analysis of the human MutLalpha.MutSalpha complex

Human DNA mismatch repair is initiated by MutSalpha which ATP-dependently recruits MutLalpha. Analysis of this complex is difficult due to its transient and dynamic nature. We have optimized conditions for investigation of MutLalpha.MutSalpha complexes using a DNA pulldown assay. Non-specific DNA end-binding, which frequently interfered with analysis of the interaction, did not occur under the applied conditions. MutSalpha had significantly higher affinity to DNA mispairs, but its interaction with MutLalpha did not require a mismatch. Complex formation was best supported by low magnesium concentration and low temperature at physiological pH and salt concentration. Complex formation was delayed by the slowly hydrolyzable ATP analog ATPgammaS, undetectable with the non-hydrolyzable analog AMP-PNP, and occurred weakly with a combination of AMP-PNP and ADP, confirming that hydrolysis was required. The described conditions likely capture an intermediate of the repair reaction which has bound ATP and ADP in the two nucleotide-binding sites of MutSalpha.     

Structure and function of the N-terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase

Human MutLalpha, a heterodimer of hMLH1 and hPMS2, is essential for DNA mismatch repair. Inactivation of the hmlh1 or hpms2 genes by mutation or epigenesis causes genomic instability and a predisposition to hereditary non-polyposis cancer. We report here the X-ray crystal structures of the conserved N-terminal 40 kDa fragment of hPMS2, NhPMS2, and its complexes with ATPgammaS and ADP at 1.95, 2.7 and 2.7 A resolution, respectively. The NhPMS2 structures closely resemble the ATPase fragment of Escherichia coli MutL, which coordinates protein-protein interactions in mismatch repair by undergoing structural transformation upon binding of ATP. Unlike the E.coli MutL, whose ATPase activity requires protein dimerization, the monomeric form of NhPMS2 is active both in ATP hydrolysis and DNA binding. NhPMS2 is the first example of a GHL ATPase active as a monomer, suggesting that its activity may be modulated by hMLH1 in MutLalpha, and vice versa. The potential heterodimer interface revealed by crystallography provides a mutagenesis target for functional studies of MutLalpha.     

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.     

Human mismatch repair: reconstitution of a nick-directed bidirectional reaction

Bidirectional mismatch repair directed by a strand break located 3' or 5' to the mispair has been reconstituted using seven purified human activities: MutSalpha, MutLalpha, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase delta. In addition to DNA polymerase delta, PCNA, RFC, and RPA, 5'-directed repair depends on MutSalpha and EXOI, whereas 3'-directed mismatch correction also requires MutLalpha. The repair reaction displays specificity for DNA polymerase delta, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase delta in the excision step of repair. RFC and PCNA dramatically activate polymerase delta-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase delta are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3'- and 5'-directed excision in HeLa nuclear extracts by only 20-30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase.     

Efficient repair of A/C mismatches in mouse cells deficient in long-patch mismatch repair

A previously unrecognized mismatch repair activity is described. Extracts of immortalized MSH2-deficient mouse fibroblasts did not correct most single base mispairs. The same extracts carried out efficient repair of A/C mismatches. A/G mispairs were less efficiently corrected and there was no significant repair of A/A. MLH1-defective mouse extracts also repaired an A/C mispair. A/C correction by Msh2(-/-) mouse cell extracts was not affected by antibodies against the PMS2 protein, which inhibited long-patch mismatch repair. A/C repair activity is thus independent of MutSalpha, MutSbeta and MutLalpha. A/C mismatches were corrected 5-fold more efficiently by extracts of Msh2 knockout mouse cells than by comparable extracts prepared from hMSH2- or hMLH1-deficient human cells. MSH2-independent A/C correction by mouse cell extracts did not require a nick in the circular duplex DNA substrate. Repair involved replacement of the A and was associated with the resynthesis of a limited stretch of 相似文献   

Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades

Mismatch-repair (MMR) systems promote genomic stability by correction of DNA replication errors. Thus, MMR proteins--prokaryotic MutS and MutL homodimers or their MutSalpha and MutLalpha heterodimer homologs, plus accessory proteins--specifically couple mismatch recognition to nascent-DNA excision. In vivo excision-initiation signals--specific nicks in some prokaryotes, perhaps growing 3' ends or Okazaki-fragment 5' ends in eukaryotes--are efficiently mimicked in vitro by nicks or gaps in exogenous DNA substrates. In some models for recognition-excision coupling, MutSalpha bound to mismatches is induced by ATP hydrolysis, or simply by binding of ATP, to slide along DNA to excision-initiation sites, perhaps in association with MutLalpha and accessory proteins. In other models, MutSalpha.MutLalpha complexes remain fixed at mismatches and contact distant excision sites by DNA looping. To challenge the hypothesis that recognition complexes remain fixed, we placed biotin-streptavidin blockades between mismatches and pre-existing nicks. In human nuclear extracts, mismatch efficiently provoked the initiation of excision despite the intervening barriers, as predicted. However, excision progress and therefore mismatch correction were prevented.     

Mutations affecting a putative MutLalpha endonuclease motif impact multiple mismatch repair functions

Mutations in DNA mismatch repair (MMR) lead to increased mutation rates and higher recombination between similar, but not identical sequences, as well as resistance to certain DNA methylating agents. Recently, a component of human MMR machinery, MutLalpha, has been shown to display a latent endonuclease activity. The endonuclease active site appears to include a conserved motif, DQHA(X)(2)E(X)(4)E, within the COOH-terminus of human PMS2. Substitution of the glutamic acid residue (E705) abolished the endonuclease activity and mismatch-dependent excision in vitro. Previously, we showed that the PMS2-E705K mutation and the corresponding mutation in Saccharomyces cerevisiae were both recessive loss of function alleles for mutation avoidance in vivo. Here, we show that mutations impacting this endonuclease motif also significantly affect MMR-dependent suppression of homeologous recombination in yeast and responses to S(n)1-type methylating agents in both yeast and mammalian cells. Thus, our in vivo results suggest that the endonuclease activity of MutLalpha is important not only in MMR-dependent mutation avoidance but also for recombination and damage response functions.     

Mapping the dimer interface in the C-terminal domains of the yeast MLH1-PMS1 heterodimer

Yeast MutLalpha is a heterodimer of MLH1 and PMS1 that participates in a variety of DNA transactions, including DNA mismatch repair. Formation of the MutLalpha heterodimer requires that the C-terminal domains of MLH1 and PMS1 interact in a manner that is not yet fully understood. Here we investigate the interactions involved in heterodimerization. Using protein surface modification and mass spectrometry, we identify numerous lysine residues that are exposed to solvent in monomeric MLH1. A corresponding analysis of the MLH1-PMS1 heterodimer reveals that three of these exposed residues, K665, K675, and K704, are no longer solvent accessible in the heterodimer, suggesting that they are within the dimer interface. We refine secondary structure predictions and sequence alignments of C-terminal residues of seven eukaryotic MutL homologues and then develop homology models for the N- and C-terminal domains of MLH1. On the basis of this information, we present a model for interaction of the C-terminal domains of MLH1 and PMS1.     

The FANCJ/MutLalpha interaction is required for correction of the cross-link response in FA-J cells

FANCJ also called BACH1/BRIP1 was first linked to hereditary breast cancer through its direct interaction with BRCA1. FANCJ was also recently identified as a Fanconi anemia (FA) gene product, establishing FANCJ as an essential tumor suppressor. Similar to other FA cells, FANCJ-null (FA-J) cells accumulate 4N DNA content in response to DNA interstrand crosslinks (ICLs). This accumulation is corrected by reintroduction of wild-type FANCJ. Here, we show that FANCJ interacts with the mismatch repair complex MutLalpha, composed of PMS2 and MLH1. Specifically, FANCJ directly interacts with MLH1 independent of BRCA1, through its helicase domain. Genetic studies reveal that FANCJ helicase activity and MLH1 binding, but not BRCA1 binding, are essential to correct the FA-J cells' ICL-induced 4N DNA accumulation and sensitivity to ICLs. These results suggest that the FANCJ/MutLalpha interaction, but not FANCJ/BRCA1 interaction, is essential for establishment of a normal ICL-induced response. The functional role of the FANCJ/MutLalpha complex demonstrates a novel link between FA and MMR, and predicts a broader role for FANCJ in DNA damage signaling independent of BRCA1.     

Mechanisms and functions of DNA mismatch repair

DNA mismatch repair （MMR） is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including hereditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.     

Apoptosis and mutation in the murine small intestine: loss of Mlh1- and Pms2-dependent apoptosis leads to increased mutation in vivo

The mismatch repair (MMR) protein Msh2 has been shown to function in the apoptotic response to alkylating agents in vivo. Here, we extend these studies to the MutL homologues (MLH) Mlh1 and Pms2 by analysing the apoptotic response within the small intestine of gene targeted strains. We demonstrate significant differences between Msh2, Mlh1 and Pms2 mutations in influencing apoptotic signalling following 50mg/kg N-methyl-nitrosourea (NMNU), with no obvious reliance upon either Mlh1 or Pms2. However, following exposure to 100mg/kg temozolomide or lower levels of NMNU (10mg/kg) both Mlh1- and Pms2-dependent apoptosis was observed, indicating that the apoptotic response at these levels of DNA damage is dependent on the MutL homologues. Given our ability to observe a MutLalpha dependence of the apoptotic response, we tested whether perturbations of this response directly translate into increases in mutation frequency in vivo. We show that treatment with temozolomide or 10mg/kg NMNU significantly increases mutation in both the Mlh1 and Pms2 mutant mice. At higher levels of NMNU, where the apoptotic response is independent of Mlh1 and Pms2, no gene dependent increase in mutation frequency was observed. These results argue that the MutSalpha and MutLalpha are not equally important in their ability to signal apoptosis. However, when MMR does mediate apoptosis, perturbation of this response leads to long-term persistence of mutant cells in vivo.     

Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair

Expansion of triplex-forming GAA/TTC repeats in the first intron of FXN gene results in Friedreich's ataxia. Besides FXN, there are a number of other polymorphic GAA/TTC loci in the human genome where the size variations thus far have been considered to be a neutral event. Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin, which correlates with their propensity to adopt triplex structure and to block replication progression. We show that fragility is mediated by mismatch repair machinery and requires the MutSbeta and endonuclease activity of MutLalpha. We suggest that the mechanism of GAA/TTC-induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts.     

DNA mismatch repair and mutation avoidance pathways

Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2-MSH6 (MutSalpha) and MSH2-MSH3 (MutSbeta). Interestingly, MSH3 (and thus MutSbeta) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1-PMS1 (MutLalpha) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases delta and epsilon. MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR.     

Different frameshift mutation spectra in non-repetitive DNA of MutSalpha- and MutLalpha-deficient fission yeast cells

A frameshift reversion assay has been established for Schizosaccharomyces pombe, which allows detection of deletions and insertions of nucleotides in a non-repetitive DNA sequence. Compared to wild type, frameshift mutation rates were increased in the mismatch repair (MMR) mutants msh2, msh6, mlh1, and pms1, but not in a swi4 strain (defective in the Msh3 homologue). Rates were also elevated in the DNA nuclease-deficient strains rad2 (defective in the FEN-1 homologue) and exo1. In MutSalpha-deficient strains, msh2 and msh6, most of the reversions were 1bp deletions. In contrast, mlh1 and pms1 mutants, defective in MutLalpha, accumulated significantly more 2bp insertions, preferentially of the type CG to (CG)(2). Such duplications were less frequent in double mutants additionally defective in msh2, msh6, rad2, or exo1. Thus, accumulation of (CG)(2) in MutLalpha-deficient strains depends on the presence of MutSalpha, Rad2 and Exo1.