Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

phiX174 DNA-dependent DNA synthesis is catalyzed in vitro by the combination of at least 11 purified protein fractions: dnaB, dnaC(D), and dnaG gene products, DNA polymerase III, DNA elongation factors I and II, DNA binding protein, and replication factors W, X, Y, and Z. The reaction requires ATP, 4 dNTPs, and Mg+2 and is specific for phiX174 (or phiXahb) DNA. Purified replication factor Y contains phiX174 (or phiXahb) DNA-dependent ATPase (or dATPase) activity. The ATPase activity is poorly stimulated by other single-stranded DNA, by double-stranded DNA, or by RNA. The products of the phiX174 DNA-dependent ATPase activity of factor Y are Pi and ADP (or dADP). The association of phiX174 DNA-dependent ATPase activity with factor Y was shown in the following ways: (a) the two activities copurified with a constant ratio; (b) they comigrated on native polyacrylamide gel electrophoresis; (c) both activities were heat-inactivated at the same rate; and (d) both showed identical patterns of N-ethylmaleimide sensitivity.
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PMID:Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA. 12 75

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.
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PMID:A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication. 13 39

Hydrolysis of ATP by rep protein proceeds in the presence of a single-stranded region of DNA 4 residues long, but the true effector for rep ATPase appears to be a replicating fork rather than a random coil. At or near a fork in duplex DNA, rep ATPase action is different from what it is on DNA lacking secondary structure (single-stranded): (i) Km for ATP is lower, (ii) specificity is for ATP and dATP with no action on other nucleoside triphosphates, (iii) sensitivity to certain ATP analogs is reduced, (iv) presence of a DNA-nicking enzyme (e.g. cistron A protein induced by phiX174) is required, and (v) Escherichia coli DNA binding protein facilitates rather than inhibits. During the separation of strands accompanying replication, 2 molecules of nucleoside triphosphate (ATP or dATP) are hydrolyzed for every nucleotide polymerized. Utilization of ATP by rep protein may provide energy for catalytic strand separation at a fork in advance of replication.
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PMID:ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork. 14 74

We have isolated a new DNA-dependent ATPase from E. coli. The enzyme has been purified to greater than 90% purity. It appears to be composed of two identical polypeptide chains of molecular weight 20,000. The enzyme catalyzed the hydrolysis of ATP in the presence, but not in the absence, of single-stranded DNA. Double-stranded DNA is not a cofactor. The products of hydrolysis are ADP and Pi. The enzyme also catalyzed strand separation of duplex DNA in the presence of ATP and E. coli DNA binding protein. Two E. coli proteins capable of promoting strand separation have been reported previously and have been termed helicase I and II (Abdel-Monem, M., and Hoffmann-Berling, H. (1977) Eur. J. Biochem. 79, 33-38). Accordingly, this protein is named helicase III.
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PMID:Enzyme-catalyzed DNA unwinding. A DNA-dependent ATPase from E. coli. 22 86

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes (phi X174, fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, lambda p rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with lambda p rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of phi X174 replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from phi X-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.
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PMID:The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III. 38 40

A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K(+)-ATPase alpha subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor).
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PMID:Sequence motif in control regions of the H+/K+ ATPase alpha and beta subunit genes recognized by gastric specific nuclear protein(s). 131 19

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).
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PMID:In vitro replication of DNA containing either the SV40 or the polyoma origin. 289 81

The primary amino acid sequences of proteins that are receptors for estrogen, glucocorticoids, and ouabain were compared with each other using computer programs designed to detect and quantify similarities between proteins. Three regions of similarity between the estrogen receptor (ER) and the glucocorticoid receptor (GR) were identified. On the ER, residues 173-250, 323-395, and 426-458 are similar to residues 409-486, 540-612, and 644-676, respectively, on the GR. The ALIGN computer analysis of these segments on the ER and the GR gave comparison scores that were 16.8, 13.7, and 6.8 standard deviations higher, respectively, than that obtained with a comparison of randomized sequences of these proteins. The probability of getting these scores by chance is less than 10(-60), 10(-40), and 10(-11), respectively. Others have proposed that the segment on the ER and GR that is nearest their amino terminus (e.g. residues 173-250 of the ER) is part of their DNA binding domain and that the other two similar segments on each receptor, which are closer to their carboxy terminus, are part of their steroid binding domain. Here, we present evidence to support both of these hypotheses. First, an Align computer analysis indicates that residues 323-395 of the ER and residues 570-612 of the GR contain a region that is similar to a part of the alpha-subunit of the (Na+ + K+)ATPase that is hypothesized to bind the steroid ouabain. This similarity provides additional support for the proposed location of the steroid binding site on the ER, GR, and (Na+ + K+)ATPase. Second, a computer search of the protein sequence database revealed that protamine, a DNA binding protein, has some similarity to residues 255-281 of the ER, which are thought to be part of the DNA binding domain in the ER. Further, we find that residues 276-281 of the ER contain a structure that has been found at the nucleotide binding domain of some protein kinases. If this region on the ER binds ATP, then it may be involved in phosphorylation/dephosphorylation of the ER, which is thought to be important in its mechanism of action.
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PMID:Computer-based search for steroid and DNA binding sites on estrogen and glucocorticoid receptors. 302 Nov 26

The simian virus 40 (SV40) large T antigen (large tumor antigen), in conjunction with a topoisomerase, a DNA binding protein, and ATP, catalyzed the conversion of a circular duplex DNA molecule containing the SV40 origin of replication to a form with unusual electrophoretic mobility that we have named form U. Analysis of this molecule revealed it to be a highly underwound covalently closed circle. DNA unwinding was not detected with DNA containing a SV40 T-antigen binding site II mutation that renders the DNA inactive in replication. The unwinding reaction requires the action of a helicase, and SV40 T-antigen preparations contain such an activity. The T-antigen-associated ability to unwind DNA copurified with other activities intrinsic to T antigen [ability to support replication of SV40 DNA containing the SV40 origin, poly(dT)-stimulated ATPase activity, and DNA helicase]. However, in contrast to the unwinding activity, the SV40 T-antigen-associated helicase activity was not sequence-specific. A variety of labeled oligonucleotides hybridized with circular single-stranded DNA were displaced by T antigen in the presence of ATP.
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PMID:Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. 302 51

The role of phosphorylation in regulating the biochemical properties of SV40 large T antigen has been examined. Treatment of purified T antigen with calf intestinal alkaline phosphatase resulted in the removal of 80% of the 32P label. This partially dephosphorylated T antigen displayed an increase in its ability to support DNA replication in vitro. This increase in replication activity was paralleled by an activation of specific DNA binding to site II, a necessary element within the origin of SV40 DNA replication. In contrast, the ATPase activity of dephosphorylated T antigen remained unchanged. These results demonstrate that DNA replication is regulated by phosphorylation of an origin specific DNA binding protein.
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PMID:Regulation of SV40 DNA replication by phosphorylation of T antigen. 303 73


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