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)

Clones for DNA topoisomerase IIalpha and beta (topo-IIalpha and beta) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIalpha and beta were about 80% identical for the N-terminal ATPase domain and the central core domain but only 37% for the C-terminal domain. Polyclonal antibodies were raised against C-terminal polypeptides specific to topo-IIalpha and beta. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIalpha was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2'-deoxyuridine, whereas topo-IIbeta was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIalpha and beta suggested that a sequence region (residues 1280-1294) in the C-terminal domain of topo-IIalpha was effective in co-localization with sites of DNA replication. This region consists of a QTxhxF motif (x, any residue; h, hydrophobic residue) followed by a KR-rich sequence, which resembles those found in several proteins known to associate with proliferating cell nuclear antigen (PCNA) or targeted to the replication factory. An in vitro pull-down assay with glutathione-S-transferase-PCNA and (His)6-tagged truncated forms of topo-IIalpha demonstrated that polypeptides containing the above region (residues 1158-1553 or 1158-1294) bound to PCNA in vitro.
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PMID:Co-localization of chicken DNA topoisomerase IIalpha, but not beta, with sites of DNA replication and possible involvement of a C-terminal region of alpha through its binding to PCNA. 1145 53

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.
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PMID:A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability. 1145 65

We have determined the nucleotide sequences of about 55% of the region of the DNA topoisomerase II gene (approximately 2.3 kb) isolated from the pathogenic Candida species, C. dubliniensis, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, C. guilliermondii and C. lusitaniae. Evolutionary relationships among nine Candida species including those of C. albicans and C. glabrata were studied based on the DNA topoisomerase II gene. The nucleotide sequences of 2192 bp, which covered two catalytic domains, ATPase and cutting/resealing, were subjected to phylogenetic analysis. Sequence comparison and evolutionary analysis have revealed that the Candida species tested here are not monophyletic, and the two strains within the species C. tropicalis and C. parapsilosis are too diverse to be in a single species. A wide variety of divergence was observed among the functional domains of DNA topoisomerase II, suggesting that Candida species were in different evolutionary paths at least as regarding the DNA topoisomerase II gene. Sequence information and the observation on the species-specific manner of molecular evolution of DNA topoisomerase II in Candida will be applied to develop a method of identification and characterization of the Candida species in both natural and clinical isolates.
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PMID:Phylogenetic relationship and mode of evolution of yeast DNA topoisomerase II gene in the pathogenic Candida species. 1147 May 34

We have prepared full-length Drosophila and human topoisomerase II and truncation constructs containing the amino-terminal ATPase domain, and we have analyzed their biochemical properties. The ATPase activity of the truncation proteins, similar to that of the full-length proteins, is greatly stimulated by the presence of DNA. This activity of the truncation proteins is also sensitive to the inhibition by the drug bisdioxopiperazine, ICRF-193, albeit at a much lower level than the full-length protein. Therefore, bisdioxopiperazine can directly interact with the NH(2)-terminal ATPase domain, but the drug-enzyme interaction may involve other domains as well. The ATPase activity of the ATPase domain protein showed a quadratic dependence on enzyme concentration, suggesting that dimerization of the NH(2)-terminal domain is a rate-limiting step. Using both protein cross-linking and sedimentation equilibrium analysis, we showed that the ATPase domain exists as a monomer in the absence of cofactors but can readily dimerize in the presence of a nonhydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate. More interestingly, both ATP and ADP can also promote protein dimerization. This result thus suggests that the protein clamp, mediated through the dimerization of ATPase domain, remains closed after ATP hydrolysis and opens upon the dissociation of ADP.
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PMID:ATPase domain of eukaryotic DNA topoisomerase II. Inhibition of ATPase activity by the anti-cancer drug bisdioxopiperazine and ATP/ADP-induced dimerization. 1185 Apr 31

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.
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PMID:Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha. 1204 90

Reverse gyrase, the only topoisomerase known to positively supercoil DNA, has an N-terminal ATPase domain that drives the activity of a topoisomerase domain. This study shows that the N-terminal domain represses topoisomerase activity in the absence of nucleotide, and nucleotide binding is sufficient to relieve the repression. A "latch" region in the N-terminal part was observed to close over the topoisomerase domain in the reverse gyrase crystal structure. Mutants lacking all or part of the latch relax DNA in the absence of nucleotide, indicating that this region mediates topoisomerase repression. The mutants also show altered DNA-dependent ATPase activity, suggesting that the latch may be involved in coupling nucleotide hydrolysis to supercoiling. It is not required for this process, however, because the mutants can still positively supercoil DNA. Nucleotide hydrolysis is essential to the specificity of reverse gyrase for increasing the linking number of DNA. Although with ATP the enzyme performs strand passage always toward increasing linking number, it can increase or decrease the linking number in the presence of a nonhydrolyzable ATP analog. This suggests that the mechanism of reverse gyrase is best described by a combination of recently proposed models.
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PMID:Studies of a positive supercoiling machine. Nucleotide hydrolysis and a multifunctional "latch" in the mechanism of reverse gyrase. 1204 89

We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction.
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PMID:The ATP-operated clamp of human DNA topoisomerase IIalpha: hyperstimulation of ATPase by "piggy-back" binding. 1207 77

We have isolated two overlapping genomic clones that contain the 5'-terminal portion of the human vacuolar H(+)-ATPase c subunit (ATP6L) gene. The sequence preceding the transcription initiation site, which is GC-rich, contains four GC boxes and one Oct1-binding site, but there is no TATA box or CCAAT box. In vivo footprint analysis in human cancer cells shows that two GC boxes and the Oct1-binding site are occupied by Sp1 and Oct1, respectively. We show here that treatment with anticancer agents enhances ATP6L expression. Although cisplatin did not induce ATP6L promoter activity, it altered ATP6L mRNA stability. On the other hand, the DNA topoisomerase II inhibitor, TAS-103, strongly induced promoter activity, and this effect was completely eradicated when a mutation was introduced into the Oct1-binding site. Treatment with TAS-103 increased the levels of both Sp1/Sp3 and Oct1 in nuclear extracts. Cooperative binding of Sp1 and Oct1 to the promoter is required for promoter activation by TAS-103. Incubation of a labeled oligonucleotide probe encompassing the -73/-68 GC box and -64/-57 Oct1-binding site with a nuclear extract from drug-treated KB cells yielded higher levels of the specific DNA-protein complex than an extract of untreated cells. Thus, the two transcription factors, Sp1 and Oct1 interact, in an adaptive response to DNA damage, by up-regulating expression of the vacuolar H(+)-ATPase genes. Furthermore, combination of the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A1, with TAS-103 enhanced apoptosis of KB cells with an associated increase in caspase-3 activity. Our data suggest that the induction of V-ATPase expression is an anti-apoptotic defense, and V-ATPase inhibitors in combination with low-dose anticancer agents may provide a new therapeutic approach.
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PMID:Enhanced expression of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents. 1213 27

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIbeta. We show that topo IIbeta is a DNA-dependent ATPase that appears to fit Michaelis-Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k(cat) for ATP hydrolysis by human DNA topo IIbeta in the presence of DNA is 2.25 s(-1). We have characterised a topo IIbeta derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s(-1), while not significantly altering the apparent K(m). The specificity constant for the interaction between ATP and topo IIbeta (kcat/K(mapp)) showed a 90% reduction for betaS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIbeta. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.
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PMID:Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability. 1249 Jul 10

Type IIA and type IIB topoisomerases each possess the ability to pass one DNA duplex through another in an ATP-dependent manner. The role of ATP in the strand passage reaction is poorly understood, particularly for the type IIB (topoisomerase VI) family. We have solved the structure of the ATP-binding subunit of topoisomerase VI (topoVI-B) in two states: an unliganded monomer and a nucleotide-bound dimer. We find that topoVI-B is highly structurally homologous to the entire 40-43 kDa ATPase region of type IIA topoisomerases and MutL proteins. Nucleotide binding to topoVI-B leads to dimerization of the protein and causes dramatic conformational changes within each protomer. Our data demonstrate that type IIA and type IIB topoisomerases have descended from a common ancestor and reveal how ATP turnover generates structural signals in the reactions of both type II topoisomerase families. When combined with the structure of the A subunit to create a picture of the intact topoisomerase VI holoenzyme, the ATP-driven motions of topoVI-B reveal a simple mechanism for strand passage by the type IIB topoisomerases.
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PMID:Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution. 1250 93


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