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)

Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha-32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.
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PMID:Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen. 795 65

TrwC is an essential protein in conjugative DNA transfer of the broad-host-range plasmid R388. TrwC was purified in two chromatographic steps from TrwC-overproducing bacteria. The purification procedure resulted in > 90% pure TrwC protein, which was free of contaminating nuclease activities. TrwC behaved as a dimer in gel-filtration chromatography in the presence of 550 mM NaCl, and had a pI of 10.1. The purified protein showed in-vitro ssDNA-dependent nucleoside-5'-triphosphatase and DNA helicase activities. ATP was the preferred substrate for the NTP hydrolysis reaction, which required Mg2+. The helicase activity was dependent on ATP and Mg2+. The efficiency of the unwinding reaction catalyzed by TrwC ranged from > 90% of fragment displaced for a 93-nucleotide sequence to < 5% for a 365-nucleotide sequence. Unwinding was unidirectional in the 5' to 3' direction. The enzyme turned over very slowly from one DNA substrate molecule to another. TrwC is only the second DNA helicase to be described which is involved in conjugative DNA transfer. The biochemical properties of TrwC described here confirm its functional relatedness to helicase I (TraI) encoded by plasmid F of E. coli.
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PMID:Purification and biochemical characterization of TrwC, the helicase involved in plasmid R388 conjugal DNA transfer. 800 58

Escherichia coli Rho factor is required for termination of transcription at certain sites by RNA polymerase. Binding to unstructured cytosine-containing RNA target sites, subsequent RNA-dependent ATP hydrolysis, and an RNA-DNA helicase activity that presumably facilitates termination, are considered essential for Rho function. Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination function in vitro and in vivo have been largely undefined. Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure. These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho. Models for Rho function are considered in the light of these recent revelations.
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PMID:Rho and RNA: models for recognition and response. 802 88

DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks designated Q1 to Q5 by FPLC Mono Q column chromatography. In our previous study, we observed that crude extracts prepared from xeroderma pigmentosum complementation group C (XP-C) cells contained no DNA-dependent ATPase activity at the peak position of Q1 and exhibited a broader peak with higher activity than normal Q2 at the peak position of Q2 [Yanagisawa, J., Seki, M., Ui, M., & Enomoto, T. (1992) J. Biol. Chem. 267, 3585-3588]. We have purified two DNA-dependent ATPases Q1 and Q2 from HeLa cells and characterized their properties in order to obtain a means to discriminate ATPase Q1 from Q2 in XP-C cells. The apparent molecular masses of Q1 and Q2 on SDS-polyacrylamide gel electrophoresis were 73 and 100 kDa, respectively. The two enzymes required a divalent cation for activity. DNA-dependent ATPase Q1 hydrolyzed ATP and dATP and Q2 hydrolyzed ATP preferentially among the nucleotides tested. Both enzymes preferred single-stranded DNA as a cofactor. The DNA-dependent ATPase activity of Q2 was inhibited by 90% in the presence of 200 mM NaCl, whereas that of Q1 was not affected by NaCl at concentrations up to 200 mM. Both enzymes had DNA helicase activity, that of Q1 being more resistant to NaCl than that of Q2. The DNA helicase activity of Q2 was about 150-fold higher than that of Q1, when compared with units of ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification of two DNA-dependent adenosinetriphosphatases having DNA helicase activity from HeLa cells and comparison of the properties of the two enzymes. 805 67

The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP. To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced. The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein. This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases. RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions. However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction. RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor. These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.
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PMID:A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions. 812 66

A novel DNA helicase isolated from Xenopus laevis ovaries [Poll, E. H. A., & Benbow, R. M. (1988) Biochemistry 27, 8701-8706] was characterized biochemically. The directionality of DNA unwinding was determined to be 3' to 5'. A short 3' ssDNA tail adjacent to duplex DNA was required for DNA unwinding; the minimum length of this tail was between four and nine bases. Only short duplex DNA regions were unwound: duplex DNA of 16 base pairs was readily unwound, whereas a 26 base pair duplex was not. Longer duplex regions were unwound in the presence of Escherichia coli single-strand DNA binding protein if, in addition, the duplex region was flanked by an unpaired 3' or 5' tail and the substrate resembled a branched replicative intermediate. X. laevis DNA helicase I exhibited high affinity for ssDNA, moderate affinity for dsDNA, and no affinity for RNA. DNA unwinding activity was stimulated by monovalent cations, with an optimal concentration of 150 mM for NaCl or KCl or 125 mM for Na chi PO4 or K chi PO4. The ATP analog ATP gamma S inhibited the DNA unwinding and copurifying DNA-dependent ATPase activity, whereas AMPPCP and AMPPNP moderately inhibited DNA unwinding activity and had little effect on the copurifying DNA-dependent ATPase activity. CTP was a relatively strong inhibitor of DNA unwinding activity, but GTP, UTP, dCTP, dGTP, or TTP showed moderate or no inhibition. The copurifying DNA-dependent ATPase activity was not inhibited by CTP, GTP, UTP, dCTP, dGTP, or TTP.
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PMID:Xenopus laevis ovarian DNA helicase I: A 3' to 5' helicase that unwinds short duplexes. 814 86

New methods for the purification of highly active bacteriophage lambda terminase holoenzyme, and its individual subunits, gene products (gp) A and gpNu1, have been developed. These methods are rapid, simple, reproducible, and give high yields of unaggregated protein from small volumes of culture. The procedures involve fractionation of extracts of Escherichia coli strains harboring plasmids engineered to overproduce the respective proteins. All purified proteins exist as monomers or dimers at moderate concentrations. At concentrations where holoenzyme efficiently promotes in vitro cosN-cleavage and lambda DNA packaging, gpA displays neither of these activities unless supplemented with gpNu1 and the E. coli protein integration host factor. At high protein concentrations, however, gpA can promote cos-cleavage by itself. Although gpNu1 itself cannot promote either cosN-cleavage or DNA packaging, it does modulate these activities of gpA. GpA is a DNA-stimulated ATPase whose catalytic parameters closely resemble those of the holoenzyme. Like the holoenzyme, gpA displays a DNA helicase activity which is able to melt the annealed cosN overhangs. Certain preparations of gpA appear to undergo a time-dependent amino-terminal clipping at discrete sites even in the presence of as many as four protease inhibitors and at low temperature.
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PMID:A new procedure for the purification of the bacteriophage lambda terminase enzyme and its subunits. Properties of gene product A, the large subunit. 817 92

The human ERCC3 gene, which corrects specifically the nucleotide excision repair defect in human xeroderma pigmentosum group B and cross-complements the repair deficiency in rodent UV-sensitive mutants of group 3, encodes a presumed DNA helicase that is identical to the p89 subunit of the general transcription factor TFIIH/BTF2. To examine the significance of the postulated functional domains in ERCC3, we have introduced mutations in the ERCC3 cDNA by means of site-specific mutagenesis and have determined the repair capacity of each mutant to complement the UV-sensitive phenotype of rodent group 3 cells. A conservative substitution of arginine for the invariant lysine residue in the ATPase motif (helicase domain I), six deletion mutations in the other helicase domains, and a deletion in the potential helix-turn-helix DNA-binding motif fail to complement the ERCC3 excision repair defect of rodent group 3 mutants, which implies that the helicase domains as well as the potential DNA-binding motif are required for the repair function of ERCC3. Analysis of carboxy-terminal deletions suggests that the carboxy-terminal exon may comprise a distinct determinant for the DNA repair function. In addition, we show that a functional epitope-tagged version of ERCC3 accumulates in the nucleus. Deletion of the putative nuclear location signal impairs neither the nuclear location nor the repair function, indicating that other sequences may (also) be involved in translocation of ERCC3 to the nucleus.
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PMID:Mutational analysis of ERCC3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function. 819 50

The RAD25 gene of Saccharomyces cerevisiae functions in nucleotide excision repair of ultraviolet-damaged DNA and is also required for cell viability. The RAD25 protein shows remarkable homology to the protein encoded by the human nucleotide-excision-repair gene XPB (ERCC3), mutations in which cause the cancer-prone disease xeroderma pigmentosum and also Cockayne's syndrome. Here we purify RAD25 protein from S. cerevisiae and show that it contains single-stranded DNA-dependent ATPase and DNA helicase activities. Extract from the conditional lethal mutant rad25-ts24 exhibits a thermolabile transcriptional defect which can be corrected by the addition of RAD25 protein, indicating a direct and essential role of RAD25 in RNA polymerase II transcription. The protein encoded by the rad25799am allele is defective in DNA repair but is proficient in RNA polymerase II transcription, indicating that RAD25 DNA-repair activity is separable from its transcription function. The rad25 Arg-392 encoded product, which contains a mutation in the ATP-binding motif, is defective in RNA polymerase II transcription, suggesting that the RAD25-encoded DNA helicase functions in DNA duplex opening during transcription initiation.
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PMID:RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. 820 51

Overexpressed PIF1 DNA helicase was purified from mitochondria to near homogeneity. Its ATPase and unwinding properties were characterized. The enzyme specifically utilizes ATP (or dATP) and MgCl2 (and to a lesser extent MnCl2). ATPase activity requires single-stranded DNA as an effector, duplex DNA being 100-fold less effective. The Keff, defined as the concentration of DNA required to achieve half-maximal ATPase activity, does not depend on single-stranded DNA length. Long duplex DNAs are poorly unwound and, moreover, dilution of the enzyme and its DNA substrate in the assay decreases DNA helicase activity. These data indicate that PIF1 helicase is a distributive enzyme, frequently turning from one DNA molecule to another. When forked substrates are used, unwinding by PIF1 is markedly stimulated. The enzyme has a sedimentation coefficient of 6.5 S, suggesting that it exists as a monomer in solution.
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PMID:PIF1 DNA helicase from Saccharomyces cerevisiae. Biochemical characterization of the enzyme. 825 34


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