Human MutY homolog, a DNA glycosylase involved in base excision repair, physically and functionally interacts with mismatch repair proteins human MutS homolog 2/human MutS homolog 6.

Adenines mismatched with guanines or 7,8-dihydro-8-oxo-deoxyguanines that arise through DNA replication errors can be repaired by either base excision repair or mismatch repair. The human MutY homolog (hMYH), a DNA glycosylase, removes adenines from these mismatches. Human MutS homologs, hMSH2/hMSH6 (hMutSalpha), bind to the mismatches and initiate the repair on the daughter DNA strands. Human MYH is physically associated with hMSH2/hMSH6 via the hMSH6 subunit. The interaction of hMutSalpha and hMYH is not observed in several mismatch repair-defective cell lines. The hMutSalpha binding site is mapped to amino acid residues 232-254 of hMYH, a region conserved in the MutY family. Moreover, the binding and glycosylase activities of hMYH with an A/7,8-dihydro-8-oxo-deoxyguanine mismatch are enhanced by hMutSalpha. These results suggest that protein-protein interactions may be a means by which hMYH repair and mismatch repair cooperate in reducing replicative errors caused by oxidized bases.     

Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutSalpha is abolished by a single mutation in the hMSH6 subunit

The most abundant mismatch binding factor in human cells, hMutSalpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein. The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5)DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A., Biswas, I., Camerini-Otero, R. D., and Hsieh, P. (1997) J. Biol. Chem. 272, 23811-23817). We introduced an identical mutation into one or both subunits of hMutSalpha. The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutSalpha were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA. Thus the N-terminal portion of hMSH6 appears to impart on hMutSalpha not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA.     

Functional analysis of human MutSalpha and MutSbeta complexes in yeast.

Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches. Here we perform functional analyses of these human protein complexes in yeast. We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain. Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain. However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold. Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA. The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast. Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding. The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles.     

hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutSalpha.

In extracts of human cells, base-base mismatches and small insertion/deletion loops are bound primarily by hMutSalpha, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160). Recombinant hMutSalpha bound a G/T mismatch-containing oligonucleotide with an apparent dissociation constant Kd = 2.6 nM, while its affinity for a homoduplex substrate was >20-fold lower. In the presence of ATP, hMutSalpha dissociated from mismatched oligonucleotide substrates, and this reaction was attenuated by mutating the conserved lysine in the ATP-binding domains of hMSH6, hMSH2 or both to arginine. Surprisingly, this reaction required only ATP binding, not hydrolysis. The ATPase activity of hMutSalpha variants carrying the Lys-->Arg mutation in hMSH2 or in hMSH6 was severely affected, but these mutants were still proficient in mismatch binding and were able to complement, albeit to different extents, mismatch repair-deficient cell extracts. The mismatch binding-proficient, ATPase-deficient double mutant was inactive in the complementation assay and its presence in repair-proficient extracts was inhibitory. We conclude that although the ATPase activity of hMutSalpha is dispensible for mismatch binding, it is required for mismatch correction.     

Degadration of mismatch repair hMutSalpha heterodimer by the ubiquitin-proteasome pathway

Mismatch repair plays a critical role in genome stability. This process requires several proteins including hMSH2/hMSH6 (hMutSalpha) heterodimer involved in the first stage of the process, the mispair recognition. We previously reported that in U937 and HL-60 cell lines, hMSH2 and hMSH6 protein expression was much lower than that in HeLa and KG1a cells. Here, we showed that the decreased expression of hMutSalpha results from differences in the degradation rate of both proteins by the ubiquitin-proteasome pathway. Our data suggest that in human cell lines, ubiquitin-proteasome could play an important role in the regulation of hMutSalpha protein expression, thereby regulating mismatch repair activity.     

Mutation in the magnesium binding site of hMSH6 disables the hMutSalpha sliding clamp from translocating along DNA

In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutSalpha, a heterodimer of hMSH2 and hMSH6. In the presence of ATP and magnesium, hMutSalpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutSalpha variant, hMutSalpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with translocation. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The "translocation-prone" conformation of wild type hMutSalpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gammaS or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutSalpha in the presence and in the absence of magnesium helps explain the functional differences between hMutSalpha variants incapable of binding ATP as compared with those unable to bind the metal ion.     

Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins.

The antitumor agent cis-diamminedichloroplatinum(II) (cisplatin) introduces cytotoxic DNA damage predominantly in the form of intrastrand crosslinks between adjacent purines. Binding assays using a series of duplex oligonucleotides containing a single 1,2 diguanyl intrastrand crosslink indicate that human cell extracts contain factors that preferentially recognise this type of damage when the complementary strand contains T opposite the 3', and C opposite the 5'guanine in the crosslink. Under the conditions of the band-shift assay used, little binding is observed if the positions of the T and C are reversed in the complementary strand. Similarly, duplexes containing CC or TT opposite the crosslink are recognised relatively poorly. The binding activity is absent from extracts of the colorectal carcinoma cell lines LoVo and DLD-1 in which the hMutSalpha mismatch recognition complex is inactivated by mutation. Extensively purified human hMutSalpha exhibits the same substrate preference and binds to the mismatched platinated DNA at least as well as to an identical unplatinated duplex containing a single G.T mismatch. It is likely, therefore, that human mismatch repair may be triggered by 1,2 diguanyl intrastrand crosslinks that have undergone replicative bypass.     

Identification of mismatch repair protein complexes in HeLa nuclear extracts and their interaction with heteroduplex DNA

Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.     

Mismatch repair processing of carcinogen-DNA adducts triggers apoptosis

The DNA mismatch repair pathway is well known for its role in correcting biosynthetic errors of DNA replication. We report here a novel role for mismatch repair in signaling programmed cell death in response to DNA damage induced by chemical carcinogens. Cells proficient in mismatch repair were highly sensitive to the cytotoxic effects of chemical carcinogens, while cells defective in either human MutS or MutL homologs were relatively insensitive. Since wild-type cells but not mutant cells underwent apoptosis upon treatment with chemical carcinogens, the apoptotic response is dependent on a functional mismatch repair system. By analyzing p53 expression in several pairs of cell lines, we found that the mismatch repair-dependent apoptotic response was mediated through both p53-dependent and p53-independent pathways. In vitro biochemical studies demonstrated that the human mismatch recognition proteins hMutSalpha and hMutSbeta efficiently recognized DNA damage induced by chemical carcinogens, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. Taken together, these studies further elucidate the mechanism by which mismatch repair deficiency predisposes to cancer, i.e., the deficiency not only causes a failure to repair mismatches generated during DNA metabolism but also fails to direct damaged and mutation-prone cells to commit suicide.     

hMutSbeta is required for the recognition and uncoupling of psoralen interstrand cross-links in vitro

The removal of interstrand cross-links (ICLs) from DNA in higher eucaryotes is not well understood. Here, we show that processing of psoralen ICLs in mammalian cell extracts is dependent upon the mismatch repair complex hMutSbeta but is not dependent upon the hMutSalpha complex or hMlh1. The processing of psoralen ICLs is also dependent upon the nucleotide excision repair proteins Ercc1 and Xpf but not upon other components of the excision stage of this pathway or upon Fanconi anemia proteins. Products formed during the in vitro reaction indicated that the ICL has been removed or uncoupled from the cross-linked substrate in the mammalian cell extracts. Finally, the hMutSbeta complex is shown to specifically bind to psoralen ICLs, and this binding is stimulated by the addition of PCNA. Thus, a novel pathway for processing ICLs has been identified in mammalian cells which involves components of the mismatch repair and nucleotide excision repair pathways.     

hMSH4-hMSH5 recognizes Holliday Junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes

Five MutS homologs (MSH), which form three heterodimeric protein complexes, have been identified in eukaryotes. While the human hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers operate primarily in mitotic mismatch repair (MMR), the biochemical function(s) of the meiosis-specific hMSH4-hMSH5 heterodimer is unknown. Here, we demonstrate that purified hMSH4-hMSH5 binds uniquely to Holliday Junctions. Holliday Junctions stimulate the hMSH4-hMSH5 ATP hydrolysis (ATPase) activity, which is controlled by Holliday Junction-provoked ADP-->ATP exchange. ATP binding by hMSH4-hMSH5 induces the formation of a hydrolysis-independent sliding clamp that dissociates from the Holliday Junction crossover region, embracing two homologous duplex DNA arms. Fundamental differences between hMSH2-hMSH6 and hMSH4-hMSH5 Holliday Junction recognition are detailed. Our results support the attractive possibility that hMSH4-hMSH5 stabilizes and preserves a meiotic bimolecular double-strand break repair (DSBR) intermediate.     

Hereditary cancer-associated missense mutations in hMSH6 uncouple ATP hydrolysis from DNA mismatch binding

Hereditary nonpolyposis colorectal cancer is caused by germline mutations in DNA mismatch repair genes. The majority of cases are associated with mutations in hMSH2 or hMLH1; however, about 12% of cases are associated with alterations in hMSH6. The hMSH6 protein forms a heterodimer with hMSH2 that is capable of recognizing a DNA mismatch. The heterodimer then utilizes its adenosine nucleotide processing ability in an, as of yet, unclear mechanism to facilitate communication between the mismatch and a distant strand discrimination site. The majority of reported mutations in hMSH6 are deletions or truncations that entirely eliminate the function of the protein; however, nearly a third of the reported variations are missense mutations whose functional significance is unclear. We analyzed seven cancer-associated single amino acid alterations in hMSH6 distributed throughout the functional domains of the protein to determine their effect on the biochemical activity of the hMSH2-hMSH6 heterodimer. Five alterations affect mismatch-stimulated ATP hydrolysis activity providing functional evidence that missense variants of hMSH6 can disrupt mismatch repair function and may contribute to disease. Of the five mutants that affect mismatch-stimulated ATP hydrolysis, only two (R976H and H1248D) affect mismatch recognition. Thus, three of the mutants (G566R, V878A, and D803G) appear to uncouple the mismatch binding and ATP hydrolysis activities of the heterodimer. We also demonstrate that these three mutations alter ATP-dependent conformation changes of hMSH2-hMSH6, suggesting that cancer-associated mutations in hMSH6 can disrupt the intramolecular signaling that coordinates mismatch binding with adenosine nucleotide processing.     

Phosphorylated hMSH6: DNA mismatch versus DNA damage recognition

DNA mismatch repair (MMR) maintains genomic integrity by correction of mispaired bases and insertion-deletion loops. The MMR pathway can also trigger a DNA damage response upon binding of MutSα to specific DNA lesions such as O(6)methylguanine (O(6)meG). Limited information is available regarding cellular regulation of these two different pathways. Within this report, we demonstrate that phosphorylated hMSH6 increases in concentration in the presence of a G:T mismatch, as compared to an O(6)meG:T lesion. TPA, a kinase activator, enhances the phosphorylation of hMSH6 and binding of hMutSα to a G:T mismatch, though not to O(6)meG:T. UCN-01, a kinase inhibitor, decreases both phosphorylation of hMSH6 and binding of hMutSα to G:T and O(6)meG:T. HeLa MR cells, pretreated with UCN-01 and exposed to MNNG, undergo activation of Cdk1 and mitosis despite phosphorylation of Chk1 and inactivating phosphorylation of Cdc25c. These results indicate that UCN-01 may inhibit an alternative cell cycle arrest pathway associated with the MMR pathway that does not involve Cdc25c. In addition, recombinant hMutSα containing hMSH6 mutated at an N-terminal cluster of four phosphoserines exhibits decreased phosphorylation and decreased binding of hMutSα to G:T and O(6)meG:T. Taken together, these results suggest a model in which the amount of phosphorylated hMSH6 bound to DNA is dependent on the presence of either a DNA mismatch or DNA alkylation damage. We hypothesize that both phosphorylation of hMSH6 and total concentration of bound hMutSα are involved in cellular signaling of either DNA mismatch repair or MMR-dependent damage recognition activities.     

Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1

In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutSα) and hMSH2/hMSH3 (hMutSβ) are the mismatch recognition factors of the mismatch repair pathway. hMutSα has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutSα and hMutLα. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutSα binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutSα. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.     

The effect of O6-methylguanine DNA adducts on the adenosine nucleotide switch functions of hMSH2-hMSH6 and hMSH2-hMSH3

The human homologs of prokaryotic mismatch repair have been shown to mediate the toxicity of certain DNA damaging agents; cells deficient in the mismatch repair pathway exhibit resistance to the killing effects of several of these agents. Although previous studies have suggested that the human MutS homologs, hMSH2-hMSH6, bind to DNA containing a variety of DNA adducts, as well as mispaired nucleotides, a number of studies have suggested that DNA binding does not correlate with repair activity. In contrast, the ability to process adenosine nucleotides by MutS homologs appears to be fundamentally linked to repair activity. In this study, oligonucleotides containing a single well defined O(6)-methylguanine adduct were used to examine the extent of lesion-provoked DNA binding, single-step ADP --> ATP exchange, and steady-state ATPase activity by hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers. Interestingly, O(6)-methylguanine lesions when paired with either a C or T were found to stimulate ADP --> ATP exchange, as well as the ATPase activity of purified hMSH2-hMSH6, whereas there was no significant stimulation of hMSH2-hMSH3. These results suggest that O(6)-methylguanine uniquely activates the molecular switch functions of hMSH2-hMSH6.     

Dissociation of mismatch recognition and ATPase activity by hMSH2-hMSH3.

MSH2-MSH3 directs the repair of insertion/deletion loops of up to 13 nucleotides in vivo and in vitro. To examine the biochemical basis of this repair specificity, we characterized the mispair binding and ATPase activity of hMSH2-hMSH3. The ATPase was found to be regulated by a mismatch-stimulated ADP --> ATP exchange, which induces a conformational transition by the protein complex. We demonstrated strong binding of hMSH2-hMSH3 to an insertion/deletion loop containing 24 nucleotides that is incapable of provoking ADP --> ATP exchange, suggesting that mismatch recognition appears to be necessary but not sufficient to induce the intrinsic ATPase. These studies support the idea that hMSH2-hMSH3 functions as an adenosine nucleotide-regulated molecular switch that must be activated by mismatched nucleotides for classical mismatch repair to occur.     

hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA

Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.