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)

Various antitumor and antibacterial agents target type II DNA topoisomerases, stabilizing a cleaved DNA reaction intermediate and thereby converting topoisomerase into a cellular poison. Two 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant bacteriophage T4 topoisomerases have previously been characterized biochemically, and we have now determined the sequence of the causative mutations. In one case, a mutation (E457K) in a conserved domain of gp39 (ATPase subunit) causes resistance to antitumor agent m-AMSA but hypersensitivity to the quinolone oxolinic acid. In the second case, a combination of two amino acid substitutions (S79F and G269V) in gp52 (DNA-cleaving subunit) causes resistance to both m-AMSA and oxolinic acid. The S79F mutation is responsible for drug resistance, whereas the G269V mutation suppresses a topoisomerase deficiency caused by S79F. Surprisingly, the G269V mutation by itself causes a dramatic hypersensitivity to both inhibitors, defining a new class of topoisomerase mutants. Because S79 and the adjacent N78 are homologous to two key residues of DNA gyrase that affect quinolone sensitivity, we generated additional amino acid substitutions at these two positions. The substitutions alter sensitivity to m-AMSA and to oxolinic acid, sometimes in opposite directions. Furthermore, the quinolone sensitivities of the various mutants paralleled those of corresponding gyrase mutants. These results support models in which both quinolones and antitumor agents bind to a conserved site that overlaps the active site of the enzyme.
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PMID:Mutations of the bacteriophage T4 type II DNA topoisomerase that alter sensitivity to antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide and an antibacterial quinolone. 951 14

Substituting Lys359 with either Gln or Glu in the highly conserved QTK-loop in the DNA gyrase B protein homologous domain of Drosophila topoisomerase II inactivates its catalytic activities. Although strand passage and DNA-dependent ATPase activities are affected in these mutant proteins, their DNA cleavage activity is comparable with the wild-type enzyme and can be stimulated to the same level by topoisomerase-targeting anticancer drugs. The sequence specificity in the DNA cleavage reaction remains unaltered for the mutant proteins. We have used both glass fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both Gln and Glu mutant proteins can form a clamp complex in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, albeit with a lower efficiency than the wild-type enzyme. However, the mutant proteins can form a stable complex either in the presence of ATP or in the absence of any cofactors. These results are in an interesting contrast with the wild-type enzyme, which cannot form a stable complex under similar conditions. Our data suggest that Lys359 is critical for the catalytic activity of topoisomerase II and may have an important function in the ATP signaling process.
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PMID:Identifying Lys359 as a critical residue for the ATP-dependent reactions of Drosophila DNA topoisomerase II. 954 89

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.
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PMID:Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159. 967 16

We have constructed clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. We show that the N-terminal domain (approximately 50 kDa) has an intrinsic ATPase activity that can be stimulated by DNA. The enzyme obeys Michaelis-Menten kinetics showing a approximately 6-fold increase in kcat in the presence of DNA. Cross-linking studies indicate that the N-terminal domain is a dimer in the absence and presence of nucleotides. Using site-directed mutagenesis, we have identified the catalytic residue for ATP hydrolysis as Glu86. Phosphorylation of the N-terminal domain with protein kinase C does not affect the ATPase activity. The ATPase domain of human topoisomerase IIalpha shows significant differences from its counterpart in DNA gyrase and we discuss the mechanistic implications of these data.
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PMID:The N-terminal domain of human topoisomerase IIalpha is a DNA-dependent ATPase. 983 94

DNA topoisomerase II catalyzes two different chemical reactions as part of its DNA transport cycle: ATP hydrolysis and DNA breakage/religation. The coordination between these reactions was studied using mutants of yeast topoisomerase II that are unable to covalently cleave DNA. In the absence of DNA, the ATPase activities of these mutant enzymes are identical to the wild type activity. DNA binding stimulates the ATPase activity of the mutant enzymes, but with steady-state parameters different from those of the wild type enzyme. These differences were examined through DNA binding experiments and pre-steady-state ATPase assays. One mutant protein, Y782F, binds DNA with the same affinity as wild type protein. This mutant topologically traps one DNA circle in the presence of a nonhydrolyzable ATP analog under the same conditions that the wild type protein catenates two circles. Rapid chemical quench and pulse-chase ATPase experiments reveal that the mutant proteins bound to DNA have the same sequential hydrolysis reaction cycle as the wild type enzyme. Binding of ATP to the mutants is not notably impaired, but hydrolysis of the first ATP is slower than for the wild type enzyme. Models to explain these results in the context of the entire DNA topoisomerase II reaction cycle are discussed.
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PMID:Kinetic and thermodynamic analysis of mutant type II DNA topoisomerases that cannot covalently cleave DNA. 992 Aug 89

Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance.
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PMID:Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform. 1041 8

Collisions between replication forks and topoisomerase-drug-DNA ternary complexes result in the inhibition of DNA replication and the conversion of the normally reversible ternary complex to a nonreversible form. Ultimately, this can lead to the double strand break formation and subsequent cell death. To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes. A strand displacement assay was employed to assess the effect of topoisomerase IV (Topo IV)-norfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-antigen, and UvrD DNA helicases. The ternary complexes inhibited the strand displacement activities of these DNA helicases. Unlike replication fork arrest, however, this general inhibition of DNA helicases by Topo IV-norfloxacin-DNA ternary complexes did not require the cleavage and reunion activity of Topo IV. We also examined the reversibility of the ternary complexes after collisions with these DNA helicases. UvrD converted the ternary complex to a nonreversible form, whereas DnaB, T7 gene 4 protein, and SV40 T-antigen did not. These results suggest that the inhibition of DnaB translocation may be sufficient to arrest the replication fork progression but it is not sufficient to generate cytotoxic DNA lesion.
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PMID:Interactions between DNA helicases and frozen topoisomerase IV-quinolone-DNA ternary complexes. 1042 58

Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.
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PMID:Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO. 1044 47

Topoisomerase II-catalyzed DNA transport requires coordination between two distinct reactions: ATP hydrolysis and DNA cleavage/religation. To further understand how these reactions are coupled, inhibition by the clinically used anticancer drug etoposide was studied. The IC(50) for perturbing the DNA cleavage/religation equilibrium is nucleotide-dependent; its value is 6 microM in the presence of ATP, 25 microM in the presence of a nonhydrolyzable ATP analog, and 45 microM in the presence of ADP or no nucleotide. This inhibition was further characterized using steady-state and pre-steady-state ATPase and decatenation assays. Etoposide is a hyperbolic noncompetitive inhibitor of the ATPase activity with a K(i)(app) of 5.6 microM no inhibition of ATP hydrolysis is seen in the absence of DNA cleavage. In order to determine which steps of the ATPase mechanism etoposide inhibits, pre-steady-state analysis was performed. These results showed that etoposide does not reduce the rate of binding two ATP, hydrolyzing the first ATP, or releasing the second ADP. Inhibition is therefore associated with the first product release step or hydrolysis of the second ATP, suggesting that DNA religation normally occurs at one of these two steps. Multiple turnover decatenation is inhibited when etoposide is present; however, single turnover decatenation occurs normally. The implications of these results are discussed in terms of their contribution to our current model for the topoisomerase II mechanism.
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PMID:Yeast topoisomerase II is inhibited by etoposide after hydrolyzing the first ATP and before releasing the second ADP. 1052 57

DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme. Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC(50) of 6.5 +/- 1 microM for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP. When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP. By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP. These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.
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PMID:Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193. 1064 21


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