Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.
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PMID:DNA mismatch repair-dependent response to fluoropyrimidine-generated damage. 1561 Oct 52

Cadmium (Cd2+) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd2+ exposure on the enzymatic activity of the mismatch binding complex MSH2-MSH6. Our results indicate that Cd2+ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2-MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd2+ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd2+. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn2+) does not overcome the direct inhibitory effect of Cd2+ on the MSH2-MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd2+ was prevented by excess Zn2+, probably by blocking the entry of Cd2+ into the cell. We conclude that the inhibition of MMR by Cd2+ is through the inactivation of the ATPase activity of the MSH2-MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype.
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PMID:Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex. 1574

Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC) is a hereditary syndrome with genetic heterogeneity. The disease is caused by mutations or epigenetic silencing in DNA mismatch repair genes, MLH1, MSH2, MSH6, PMS2 and MLH3, although the vast majority of cases correspond to mutations of MLH1 and MSH2. We herein describe a nucleotide change, c.2063T>G in exon 13 of the MSH2 gene, present in families that fulfill the Amsterdam criteria for Lynch syndrome and originate from northern Tenerife (Canary Islands-Spain). This mutation is expected to result in a nonconservative amino acid change, M688R, at the ATPase domain of the MSH2 protein. We found five large families with this mutation, and about half the individuals heterozygous for M688R developed malignancies by the sixth decade of life. In many cases analyzed, their tumors revealed loss of the normal allele, being homozygous for M688R. There is an evidence of historical isolation for the population studied, which could have favored a considerable genetic drift. The presence of the same mutation and the disease associated-haplotype conservation in families not directly related can be probably the consequence of a bottleneck in the founding of this population (rather than a relatively recent founding of the mutation).
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PMID:New founding mutation in MSH2 associated with hereditary nonpolyposis colorectal cancer syndrome on the Island of Tenerife. 1650 24

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein-protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.
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PMID:Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae. 1772 Sep 36

FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.
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PMID:FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability. 1842 15

The human mismatch repair (MMR) gene MSH2 is the second most frequently mutated hereditary nonpolyposis colorectal cancer (HNPCC) susceptibility locus. Given that missense mutations account for 17% of all identified alterations in this gene, the study of their pathogenicity is of increasing importance. Previously, we showed that pathogenic MSH2 missense mutations typically impaired the repair activity of the protein. In this study, we took advantage of its crystal structure and attempted to correlate the mismatch binding and ATP-catalyzed mismatch release activities with the location of 18 nontruncating MSH2 mutations. We observed that the MMR-deficient mutations situated in the amino-terminal connector and lever domains of MSH2 (V161D, G162R, G164R, L173P, L187P, C333Y, and D603N) affected protein stability, whereas mutations in the ATPase domain (A636P, G674A, C697F, I745_I746del, and E749 K) mainly caused defects in mismatch binding or release. Of the MMR-proficient variants, four (T33P, A272 V, G322D, and V923E) showed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) protein, while two variants (N127S and A834 T) showed no defects in the assays. Similar to our biochemical data, the mutations that affected protein stability were associated with an absence of the protein in tumors in immunohistochemical (IHC) analyses. In contrast, the protein with the mutation E749 K, which abrogates MMR but not protein stability, is well expressed in tumors. In conclusion, pathogenic missense mutations in MSH2 may interfere with different mechanisms that tend to cluster in separate protein domains with varying effects on protein stability, which could be taken into account when interpreting IHC data.
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PMID:Mechanisms of pathogenicity in human MSH2 missense mutants. 1895 62

MutSalpha (MSH2/MSH6) and MutSbeta (MSH2/MSH3) are eukaryotic mismatch recognition proteins that preferentially process base-base and small insertion/deletion (ID) mispairs, respectively, despite the fact that cells contain a MutSalpha:MutSbeta ratio of 10:1. To explore the mechanism underlying the differential mismatch recognition by these two proteins, purified human MutSalpha and MutSbeta were analyzed individually and competitively for their abilities to interact with a T-G and an ID substrate. We show that MutSalpha has K(D) values of 26.5 and 38.2 nm for the G-T and ID substrates, respectively, and that MutSbeta has K(D) values of 76.5 and 23.5 nm for G-T and ID, respectively. Consistent with these results, competitive binding assays revealed the following relative binding affinities: MutSbeta-ID > MutSalpha-T-G > MutSalpha-ID >> MutSbeta-T-G. Interestingly, binding of MutSbeta to ID heteroduplexes is greatly stimulated when the MutSalpha:MutSbeta ratio is > or = 10. Distinct ATP/ADP binding and ATPase activities of MutSalpha and MutSbeta were also observed. In the absence of DNA, ADP binding and ATPase activities of MutSbeta are significantly higher than those of MutSalpha. However, interaction with DNA significantly stimulates the MutSalpha ATPase activity and reduces the MutSbeta ATPase activity, the consequence being that both proteins exhibit the same level of hydrolytic activity. We conclude that the preferential processing of base-base and ID heteroduplexes by MutSalpha and MutSbeta is determined by their significant differences in ATPase activity, ADP binding activity, and high cellular MutSalpha:MutSbeta ratio.
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PMID:Distinct nucleotide binding/hydrolysis properties and molar ratio of MutSalpha and MutSbeta determine their differential mismatch binding activities. 1922 87

Postreplication DNA mismatch repair is essential for maintaining the integrity of genomic information in prokaryotes and eukaryotes. The first step in mismatch repair is the recognition of base-base mismatches and insertions/deletions by bacterial MutS or eukaryotic MSH2-MSH6. Crystal structures of both proteins bound to mismatch DNA reveal a similar molecular architecture but provide limited insight into the detailed molecular mechanism of long-range allostery involved in mismatch recognition and repair initiation. This study describes normal-mode calculations of MutS and MSH2-MSH6 with and without DNA. The results reveal similar protein flexibilities and suggest common dynamic and functional characteristics. A strongly correlated motion is present between the lever domain and ATPase domains, which suggests a pathway for long-range allostery from the N-terminal DNA binding domain to the C-terminal ATPase domains, as indicated by experimental studies. A detailed analysis of individual low-frequency modes of both MutS and MSH2-MSH6 shows changes in the DNA-binding domains coupled to the ATPase sites, which are interpreted in the context of experimental data to arrive at a complete molecular-level mismatch recognition cycle. Distinct conformational states are proposed for DNA scanning, mismatch recognition, repair initiation, and sliding along DNA after mismatch recognition. Hypotheses based on the results presented here form the basis for further experimental and computational studies.
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PMID:Deciphering the mismatch recognition cycle in MutS and MSH2-MSH6 using normal-mode analysis. 1925 32

Myotonic dystrophy type 1 (DM1) is associated with one of the most highly unstable CTG*CAG repeat expansions. The formation of further repeat expansions in transgenic mice carrying expanded CTG*CAG tracts requires the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSbeta complex. It has been proposed that binding of MutSbeta to CAG hairpins blocks its ATPase activity compromising hairpin repair, thereby causing expansions. This would suggest that binding, but not ATP hydrolysis, by MutSbeta is critical for trinucleotide expansions. However, it is unknown if the MSH2 ATPase activity is dispensible for instability. To get insight into the mechanism by which MSH2 generates trinucleotide expansions, we crossed DM1 transgenic mice carrying a highly unstable >(CTG)(300) repeat tract with mice carrying the G674A mutation in the MSH2 ATPase domain. This mutation impairs MSH2 ATPase activity and ablates base-base MMR, but does not affect the ability of MSH2 (associated with MSH6) to bind DNA mismatches. We found that the ATPase domain mutation of MSH2 strongly affects the formation of CTG expansions and leads instead to transmitted contractions, similar to a Msh2-null or Msh3-null deficiency. While a decrease in MSH2 protein level was observed in tissues from Msh2(G674) mice, the dramatic reduction of expansions suggests that the expansion-biased trinucleotide repeat instability requires a functional MSH2 ATPase domain and probably a functional MMR system.
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PMID:MSH2 ATPase domain mutation affects CTG*CAG repeat instability in transgenic mice. 1943 5

Postreplication DNA mismatch repair is initiated by the eukaryotic protein MSH2-MSH6 or the prokaryotic protein MutS, both showing overall conserved structure and functionality. Crystal structures of MSH2-MSH6 and MutS bound to the mismatch DNA reveal a closed architecture of the clamp and the lever domains exhibiting strong contacts with the bent DNA backbone. Long molecular dynamics simulations of the human MSH2-MSH6 protein in the absence of a DNA show an altered conformation of the protein that reflects the protein's state before binding to DNA. The clamp and the lever domains of both MSH6 and MSH2 open in an asymmetric and dramatic fashion. The opening of the clamp and the lever domains in the absence of DNA is coupled to changes in the ATPase domains, which explains the experimentally observed diminished ATPase activity in DNA-free MSH2-MSH6 and illustrates the allosteric coupling between DNA binding and ATPase activity.
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PMID:Conformational change in MSH2-MSH6 upon binding DNA coupled to ATPase activity. 1948 59


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