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 is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP. PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A(2)B(2) gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.
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PMID:Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance. 1260 39

To better understand the contributions that the structural maintenance of chromosome proteins (SMCs) make to condensin activity, we have tested a number of biochemical, biophysical, and DNA-associated attributes of the Smc2p-Smc4p pair from budding yeast. Smc2p and Smc4p form a stable heterodimer, the "Smc2/4 complex," which upon analysis by sedimentation equilibrium appears to reversibly self-associate to form heterotetramers. Individually, neither Smc2p nor Smc4p hydrolyzes ATP; however, ATPase activity is recovered by equal molar mixing of both purified proteins. Hydrolysis activity is unaffected by the presence of DNA. Smc2/4 binds both linearized and circular plasmids, and the binding appears to be independent of adenylate nucleotide. High mole ratios of Smc2/4 to plasmid promote a geometric change in circular DNA that can be trapped as knots by type II topoisomerases but not as supercoils by a type I topoisomerase. Binding titration analyses reveal that two Smc2/4-DNA-bound states exist, one disrupted by and one resistant to salt challenge. Competition-displacement experiments show that Smc2/4-DNA-bound species formed at even high protein to DNA mole ratios remain reversible. Surprisingly, only linear and supercoiled DNA, not nicked-circular DNA, can completely displace Smc2/4 prebound to a labeled, nicked-circular DNA. To explain this geometry-dependent competition, we present two models of DNA binding by SMCs in which two DNA duplexes are captured within the inter-coil space of an Smc2/4 heterodimer. Based on these models, we propose a DNA displacement mechanism to explain how differences in geometry could affect the competitive potential of DNA.
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PMID:Biochemical analysis of the yeast condensin Smc2/4 complex: an ATPase that promotes knotting of circular DNA. 1271 26

Reverse gyrase is the only topoisomerase known to positively supercoil DNA and the only protein unique to hyperthermophiles. The enzyme comprises an N-terminal ATPase domain and a C-terminal topoisomerase I domain, which interact to couple the hydrolysis of ATP to the overwinding of DNA. The part of the ATPase domain termed the "latch" represses topoisomerase activity in the absence of nucleotide. Here I provide evidence that the latch, in addition to its regulatory role, participates in the supercoiling mechanism during the DNA cleavage and religation steps. The latch also contributes to the coordination of ATP hydrolysis and positive supercoiling by inhibiting ATPase activity in the absence of supercoiling. The latch therefore plays an important role in the communication between the two domains of reverse gyrase.
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PMID:Investigating the role of the latch in the positive supercoiling mechanism of reverse gyrase. 1275 1

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.
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PMID:Detailed in vitro pharmacological analysis of FK506-induced neuroprotection. 1287 56

We report a novel nucleolar interaction between the AAA ATPase p97/VCP and the Werner protein (WRNp), a member of the RecQ helicase family. p97/VCP mediates several important cellular functions in eucaryotic cells, including membrane fusion of the endoplasmic reticulum and Golgi and ubiquitin-dependent protein degradation. Mutations in the WRN gene cause Werner syndrome, a genetic disorder characterized by premature onset of aging symptoms, a higher incidence of cancer, and a high susceptibility to DNA damage caused by topoisomerase inhibitors. We observed that both WRNp and valosin-containing protein (VCP) were present in the nucleoplasm and in nucleolar foci in mammalian cells and that WRNp and p97/VCP physically interacted in the nucleoli. Importantly, the nucleolar WRNp/VCP complex was dissociated by treatment with camptothecin, an inhibitor of topoisomerase I, whereas other WRNp-associated protein complexes, such as WRNp/Ku 80, were not dissociated by this drug. Because WRN syndrome cells are sensitive to topoisomerase inhibitors, these observations suggest that the VCP/WRNp interaction plays an important role in WRN biology. We propose a novel role for VCP in the DNA damage response pathway through modulation of WRNp availability.
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PMID:DNA damage modulates nucleolar interaction of the Werner protein with the AAA ATPase p97/VCP. 1293 74

Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents. Bisdioxopiperazines are anticancer agents that associate with ATP-bound eukaryotic topoisomerase II (topo II) and convert the enzyme into an inactive, salt-stable clamp around DNA. To better understand both topo II and bisdioxopiperazine function, we determined the structures of the adenosine 5'-[beta,gamma-imino]-triphosphate-bound yeast topo II ATPase region (ScT2-ATPase) alone and complexed with the bisdioxopiperazine ICRF-187. The drug-free form of the protein is similar in overall fold to the equivalent region of bacterial gyrase but unexpectedly displays significant conformational differences. The ternary drug-bound complex reveals that ICRF-187 acts by an unusual mechanism of inhibition in which the drug does not compete for the ATP-binding pocket, but bridges and stabilizes a transient dimer interface between two ATPase protomers. Our data explain why bisdioxopiperazines target ATP-bound topo II, provide a structural rationale for the effects of certain drug-resistance mutations, and point to regions of bisdioxopiperazines that might be modified to improve or alter drug specificity.
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PMID:Structure of the topoisomerase II ATPase region and its mechanism of inhibition by the chemotherapeutic agent ICRF-187. 1296 18

DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival. There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations. However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive. Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB. Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme. Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl- N-nitro- N-nitrosoguanidine, that act independently of DNA gyrase. Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources. In contrast, GyrI is not effective against lesions induced by ultraviolet radiation. Furthermore, the expression of GyrI does not significantly alter the topology of DNA. Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged.
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PMID:Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents. 1368 98

Type IIA topoisomerases are multidomain enzymes composed of four major domains: the ATPase domain, the TOPRIM domain, the DNA-cleavage/religation domain and the C-terminal domain (CTD). Although crystal structures of the first three domains are available, the three-dimensional structure of the less-conserved CTD has yet to be determined. In order to provide a three-dimensional structure of this structurally uncharacterized region, the 36 kDa CTD of ParC protein, the DNA-cleavage/religation subunit of topoisomerase IV, from Bacillus stearothermophilus has been cloned, purified and crystallized. The crystals belonged to the trigonal space group P3(1) (or P3(2)), with unit-cell parameters a = b = 83.5, c = 45.1 A. The asymmetric unit contains one molecule and the solvent content is 51.2%. A 98.9% complete native data set has been collected from a frozen crystal to 2.0 A resolution with an overall R(merge) of 6.5%.
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PMID:Crystallization and preliminary X-ray crystallographic analysis of the C-terminal domain of ParC protein from Bacillus stearothermophilus. 1499 94

Topoisomerase IIalpha plays essential roles in chromosome segregation. However, it is not well understood how topoisomerase IIalpha exerts its function during mitosis. In this report, we find that topoisomerase IIalpha forms a multisubunit complex, named toposome, containing two ATPase/helicase proteins (RNA helicase A and RHII/Gu), one serine/threonine protein kinase (SRPK1), one HMG protein (SSRP1), and two pre-mRNA splicing factors (PRP8 and hnRNP C). Toposome separates entangled circular chromatin DNA about fourfold more efficiently than topoisomerase IIalpha. Interestingly, this decatenation reaction yields knotted circles, which are not seen in reactions provided with monomeric circular DNA. Our results also show that interaction among toposome-associated proteins is highest in G2/M phase but drastically diminishes in G1/S phase. These results suggest that toposome is a dynamic complex whose assembly or activation is subject to cell cycle regulation.
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PMID:Identification of toposome, a novel multisubunit complex containing topoisomerase IIalpha. 1503

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polymerase (E9), an essential and stoichiometric component of the processive polymerase (A20), a single-strand DNA-binding protein (I3), a type I topoisomerase (H6), the uracil DNA glycosylase (D4), a nucleic acid-independent nucleoside triphosphatase (D5), a serine/threonine protein kinase (B1), and a Holliday Junction resolvase (A22). All of these factors work in concert to faithfully duplicate the viral genome. Although a replication origin has not been defined for the poxviruses, cis-acting sequences found within the telomeric 200 bp have been implicated as necessary and sufficient for minichromosome replication. Replication occurs within cytoplasmic foci from approx 3 to 12 h postinfection. This chapter includes several methodologies to assay and quantitate replication in vivo, visualize replication foci microscopically, and test the integrity of central replication enzymes in vitro.
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PMID:Methods for analysis of poxvirus DNA replication. 1511 16


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