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)

DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle. Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids. To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues). Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process. Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover. The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E. coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center. We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule. Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis. A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma-imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure.
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PMID:Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center. 1073 94

DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase IIalpha, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated approximately 2-3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase IIalpha-mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase IIalpha and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIalpha was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIalpha to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase IIalpha. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIalpha.
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PMID:The p53 tumor suppressor stimulates the catalytic activity of human topoisomerase IIalpha by enhancing the rate of ATP hydrolysis. 1076 86

The DNA strand passage activity of eukaryotic topoisomerase II relies on a cascade of conformational changes triggered by ATP binding to the N-terminal domain of the enzyme. To investigate the interdomain communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha, we characterized a mutant enzyme that contains a deletion at the interface between the two domains, covering amino acids 350-407. The ATPase domain retained full activity with a rate of ATP hydrolysis that was severalfold higher than normal, but the ATPase activity was unaffected by DNA. The cleavage and religation activities of the enzyme were comparable with those of the wild-type enzyme both in the absence and presence of cancer chemotherapeutic agents. However, neither ATP nor a nonhydrolyzable ATP analog stimulated cleavage complex formation. Although both conserved domains retained full activity, the mutant enzyme was unable to coordinate these activities into strand passage. Our findings suggest that the normal conformational transitions occurring in the enzyme upon ATP binding are hampered or lacking in the mutant enzyme. Consistent with this hypothesis, the enzyme displayed an abnormal clamp closing activity. In summary, the region covering amino acids 350-407 in human topoisomerase IIalpha seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.
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PMID:Communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha. 1077 8

An Ehrlich ascites tumour cell line (EHR2) was selected for resistance to etoposide (VP16) by in vivo exposure to this agent. The resulting cell line (EHR2/VP16) was 114.3-, 5.7-, and 4.0-fold resistant to VP16, daunorubicin, and vincristine, respectively. The amount of salt-extractable immunoreactive topoisomerase IIalpha and beta in EHR2/VP16 was reduced by 30-40% relative to that in EHR2. The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expression of P-glycoprotein was unchanged. In EHR2/VP16, the steady-state accumulation of [(3)H]VP16 and daunorubicin was reduced by 64% and 17%, respectively, as compared with EHR2. Deprivation of energy by addition of sodium azide increased the accumulation of both drugs to the level of sensitive cells. When glycolysis was restored by the addition of glucose to EHR2/VP16 cells loaded with drug in the presence of sodium azide, extrusion of [(3)H]VP16 and daunorubicin was induced. Addition of verapamil (25 microM) decreased the efflux of daunorubicin to the level of sensitive cells, but had only a moderate effect on the efflux of [(3)H]VP16. The resistant cells showed moderate sensitisation to VP16 on treatment with verapamil, whereas cyclosporin A had no effect. Compared with that of sensitive cells, the ATPase activity of plasma membrane vesicles prepared from EHR2/VP16 cells was very low. Vanadate inhibited the ATPase activity of EHR2/VP16 microsomes with a K(i) value of 30 microM. ATPase activity was slightly stimulated by daunorubicin, whereas vinblastine, verapamil, and cyclosporin A had no effect. In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20-fold). MRP displays several points of resemblance to P-glycoprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistance to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in the cells and this decrease is abolished by omission of energy; and 3) MRP increases efflux of drug from cells. However, compared with that of P-glycoprotein-positive cells, the ATPase activity of MRP-positive cells is found to be low and not able to be stimulated by verapamil.
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PMID:Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide. 1085 30

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.
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PMID:Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone. 1085 31

Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.
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PMID:HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins. 1088 Apr 50

Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha.
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PMID:Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase IIalpha. 1095 49

Epipodophyllotoxins are effective anti-tumor drugs that inhibit eukaryotic DNA topoisomerase II by trapping the enzyme in a covalent complex with DNA. We show that both the recombinant N-terminal ATPase domain and the B'A' core domain of human topoisomerase IIalpha (htopoIIalpha) bind radiolabeled etoposide specifically, even in the absence of DNA. The addition of ATP impairs etoposide binding to the holoenzyme and the N-terminal domain, but not to the core domain. To see if this interference resembles that between novobiocin and ATP in the bacterial GyrB subunit, we modeled the structure of the N-terminal domain of htopoIIalpha and performed molecular docking analysis with etoposide. Mutagenesis of critical amino acids, predicted to stabilize the drug within the N-terminal domain, reveals a less efficient binding of etoposide to the mutated proteins as monitored by direct drug binding assays, although the binding of ATP is not affected.
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PMID:Analysis of etoposide binding to subdomains of human DNA topoisomerase II alpha in the absence of DNA. 1132 21

DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.
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PMID:The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release. 1135 71

The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent 'nucleosome remodelling' activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. We have now identified the p175 subunit of CHRAC as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1-ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16.
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PMID:Acf1, the largest subunit of CHRAC, regulates ISWI-induced nucleosome remodelling. 1144 19


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