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.     

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.     

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   

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.     

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

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.     

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.     

Nuclear translocation contributes to regulation of DNA excision repair activities

DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927–939; M. Christmann, M.T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3–34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89–108]. Protein interactions are not only important for function, but also for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import, co-import of proteins, and alternative transport pathways. Most excision repair proteins appear to contain classical NLS sequences directing their nuclear import, however, additional import mechanisms add alternative regulatory levels to protein import, indirectly affecting protein function. Protein co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair.     

Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism.

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent 'acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.     

Restoration of nuclear-import failure caused by triple A syndrome and oxidative stress

Triple A syndrome is an autosomal recessive neurological disease, mimicking motor neuron disease, and is caused by mutant ALADIN, a nuclear-pore complex component. We recently discovered that the pathogenesis involved impaired nuclear import of DNA repair proteins, including DNA ligase I and the cerebellar ataxia causative protein aprataxin. Such impairment was overcome by fusing classical nuclear localization signal (NLS) and 137-aa downstream sequence of XRCC1, designated stretched NLS (stNLS). We report here that the minimum essential sequence of stNLS (mstNLS) is residues 239-276, downsized by more than 100 aa. mstNLS enabled efficient nuclear import of DNA repair proteins in patient fibroblasts, functioned under oxidative stress, and reduced oxidative-stress-induced cell death, more effectively than stNLS. The stress-tolerability of mstNLS was also exerted in control fibroblasts and neuroblastoma cells. These findings may help develop treatments for currently intractable triple A syndrome and other oxidative-stress-related neurological diseases, and contribute to nuclear compartmentalization study.     

Stochastic Processes and Component Plasticity Governing DNA Mismatch Repair

DNA mismatch repair (MMR) is a DNA excision–resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch. Remarkably, the biophysical functions of the MLH/PMS proteins have been elusive for decades. Here we consider recent observations that have helped to define the mechanics of MLH/PMS proteins and their role in choreographing MMR. We highlight the stochastic nature of DNA interactions that have been visualized by single-molecule analysis and the plasticity of protein complexes that employ thermal diffusion to complete the progressions of MMR.     

Isolation and Characterization of Point Mutations in Mismatch Repair Genes That Destabilize Microsatellites in Yeast

The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches. In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2, MSH3, MLH1, and PMS1. Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes. One semidominant MSH2 mutation was identified. Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops.     

Conservation of functional asymmetry in the mammalian MutLα ATPase

The DNA mismatch repair (MMR) protein dimer MutLα is comprised of the MutL homologues MLH1 and PMS2, which each belong to the family of GHL ATPases. These ATPases undergo functionally important conformational changes, including dimerization of the NH₂-termini associated with ATP binding and hydrolysis. Previous studies in yeast and biochemical studies with the mammalian proteins established the importance of the MutLα ATPase for overall MMR function. Additionally, the studies in yeast demonstrated a functional asymmetry between the contributions of the Mlh1 and Pms1 ATPase domains to MMR that was not reflected in the biochemical studies. We investigated the effect of mutating the highly conserved ATP hydrolysis and Mg²⁺ binding residues of MLH1 and PMS2 in mammalian cell lines. Amino acid substitutions in MLH1 intended to impact either ATP binding or hydrolysis disabled MMR, as measured by instability at microsatellite sequences, to an extent similar to MLH1-null mutation. Furthermore, cells expressing these MLH1 mutations exhibited resistance to the MMR-dependent cytotoxic effect of 6-thioguanine (6-TG). In contrast, ATP hydrolysis and binding mutants of PMS2 displayed no measurable increase in microsatellite instability or resistance to 6-TG. Our findings suggest that, in vivo, the integrity of the MLH1 ATPase domain is more critical than the PMS2 ATPase domain for normal MMR functions. These in vivo results are in contrast to results obtained previously in vitro that showed no functional asymmetry within the MutLα ATPase, highlighting the differences between in vivo and in vitro systems.     

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.     

Genetic evidence for the involvement of mismatch repair proteins,PMS2 and MLH3, in a late step of homologous recombination

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3^−/− and PMS2^−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3^DN/DN and PMS2^EK/EK cells. MLH3^−/− and MLH3^DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2^−/− and PMS2^EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3^DN/DN and PMS2^EK/EK mutations had an additive effect on the heteroallelic HR. MLH3^DN/DN/PMS2^EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3^DN/DN/PMS2^EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.     

Truncation of the C-terminus of human MLH1 blocks intracellular stabilization of PMS2 and disrupts DNA mismatch repair

The human DNA mismatch repair (MMR) protein MLH1 has essential roles in the correction of replication errors and the activation of cell cycle checkpoints and cytotoxic responses to DNA damage that contribute to suppression of cancer risk. MLH1 functions as a heterodimer with the PMS2 protein, and steady state levels of PMS2 are very low in MLH1-deficient cells. Unique to MLH1 among MutL-homolog proteins, and conserved in identified eukaryotic MLH1 proteins, is the so-called C-terminal homology domain (CTH). The function of these C-terminal 20-30 amino acids is not known. We investigated the effect of a C-terminal truncation of human MLH1 (MLH1-L749X) on mammalian MMR by testing its activity in MLH1-deficient cells. We found the CTH to be essential for suppression of spontaneous mutation, activation of a cytotoxic response to 6-thioguanine, and maintenance of normal steady state levels of PMS2. Co-expression in doubly mutant Mlh1-/-; Pms2-/- fibroblasts showed that MLH1-L749X was unable to stabilize PMS2. Over-expression of MLH1-L749X did not reduce stabilization of PMS2 mediated by wild-type MLH1, indicating that truncation of the CTH reduces the ability to compete with wild-type MLH1 for interaction with PMS2. Lack of PMS2 stabilization also was observed with a previously reported pathogenic truncation (MLH1-Y750X), but not with two different point mutations in the CTH. Biochemical assays demonstrated that truncation of the CTH reduced the stability of heterodimers, although MLH1-L749X retained significant capacity for interaction with PMS2. Thus, the CTH of human MLH1 is necessary for error correction, checkpoint signaling, and for promoting interaction with, and the stability of, PMS2. Analysis of the CTH role in stabilizing PMS2 was facilitated by a novel intracellular assay for MLH1-PMS2 interaction. This assay should prove useful for identifying additional amino acids in MLH1 and PMS2 necessary for interaction in cells, and for determining the functional consequences of MLH1 mutations identified in human cancers.     

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.     

Eukaryotic DNA mismatch repair

Eukaryotic mismatch repair (MMR) has been shown to require two different heterodimeric complexes of MutS-related proteins: MSH2-MSH3 and MSH2-MSH6. These two complexes have different mispair recognition properties and different abilities to support MMR. Alternative models have been proposed for how these MSH complexes function in MMR. Two different heterodimeric complexes of MutL-related proteins, MLH1-PMS1 (human PMS2) and MLH1-MLH3 (human PMS1) also function in MMR and appear to interact with other MMR proteins including the MSH complexes and replication factors. A number of other proteins have been implicated in MMR, including DNA polymerase delta, RPA (replication protein A), PCNA (proliferating cell nuclear antigen), RFC (replication factor C), Exonuclease 1, FEN1 (RAD27) and the DNA polymerase delta and epsilon associated exonucleases. MMR proteins have also been shown to function in other types of repair and recombination that appear distinct from MMR. MMR proteins function in these processes in conjunction with components of nucleotide excision repair (NER) and, possibly, recombination.