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)

The genome of the hepatitis C virus directs the synthesis of a single polyprotein, which is proteolytically cleaved into at least nine functional proteins. The amino-terminal portion of the polyprotein forms the structural proteins, while the carboxy-terminal region constitutes a variety of viral enzymes. The nonstructural 3 (NS3) protein, consisting of amino acids 1027-1657 of the polyprotein, is believed to be a multifunctional protein with an amino-terminal serine protease domain, which is involved in polyprotein processing, and a carboxy-terminal ATPase/RNA helicase domain, presumably involved in viral replication. We have assembled an expression vector which directs the synthesis of residues 1207-1612 of the polyprotein with an amino-terminal polyhistidine purification tag. This portion of the NS3 protein contains the putative ATPase/helicase domain. The protein has been purified to yield 30-50 mg of enzymatically active protein per liter of culture. The purified NS3 protein has both NTPase and RNA helicase activities. ATP is the preferred substrate for the NTPase; GTP is also utilized; however, UTP is a very poor substrate and CTP is not utilized. The RNA helicase activity is dependent on ATP and divalent cation. Either manganese or magnesium can serve as the divalent cation.
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PMID:Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase. 748 72

mRNA degradation is an important control point in the regulation of gene expression and has been shown to be linked to the process of translation. One clear example of this linkage is the observation that nonsense mutations in a gene can accelerate the decay of the corresponding mRNA. In the yeast Saccharomyces cerevisiae, the product of the UPF1 gene, harboring zinc finger, NTP hydrolysis, and helicase motifs, was shown to be a trans-acting factor in this decay pathway. A UPF1 gene disruption results in stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. As a first step toward understanding the molecular and biochemical mechanism of nonsense-mediated mRNA decay, we have purified Upf1p from a yeast extract and characterized its nucleic acid-dependent NTPase activity, helicase activity, and nucleic acid binding properties. The results presented in this paper demonstrate that Upf1p contains both RNA- and DNA-dependent ATPase activities and RNA and DNA helicase activities. In the absence of ATP, Upf1p binds to single-stranded RNA or DNA, whereas hydrolysis of ATP facilitates its release from single-stranded nucleic acid. Based on these results, the role of Upf1p's biochemical activities in mRNA decay and translation are discussed.
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PMID:Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation. 748 20

BTF2/TFIIH from human, delta from rat, and factor b from yeast are multisubunit basal transcription factors that have been shown to be closely associated with a protein kinase capable of phosphorylating the carboxyl-terminal domain of the large subunit of RNA polymerase II (Lu, H., Zawel, L., Fischer, L., Egly, J. M., and Reinberg, D. (1992) Nature 358, 641-645; Serizawa, H., Conaway, R. C., and Conaway, J. W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7476-7480; Feaver, W. J., Gileadi, O., and Kornberg, R. D. (1991) Cell 67, 1223-1230). We report here that a DNA-dependent ATPase and the previously characterized helicase (Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J., Chambon, P., and Egly, J. M. (1993) Science 260, 58-63) are both associated with BTF2 and reside with the p89 polypeptide subunit. The DNA requirement, the effect of Sarkosyl and staurosporine inhibitors, as well as nucleotide competition experiments, clearly distinguished ATPase/helicase from the carboxyl-terminal domain kinase. Using recombinant wild type or mutated p89/ERCC3 polypeptides and different forms of DNA template, we show the connection between ATPase and the helicase.
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PMID:The DNA-dependent ATPase activity associated with the class II basic transcription factor BTF2/TFIIH. 751 95

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.
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PMID:Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody. 751 28

The open reading frame 2 (ORF2) of the potexviral genome encodes a 24- to 26-kDa protein which is part of the "triple gene block," a group of overlapping ORFs also present in the genomes of the carla-, hordei-, and furoviruses. The product of these ORFs is believed to play a role in the cell-to-cell movement of the viruses in host plants. The amino acid sequences of the homologous ORF2 products encoded by these related viruses suggest that they specify NTP binding and possibly helicase activities. We have used an Escherichia coli expression system to produce significant amounts of the 26-kDa protein (p26) encoded by foxtail mosaic potexvirus ORF2. p28 was purified to near homogeneity by conventional purification methods and some of its biochemical properties were determined. We present evidence that p26 is an ATP, CTP, and RNA binding protein with apparent ATPase activity. Western blot analysis of infected plant extracts using a polyclonal antiserum produced against p26 indicates that it is a relatively stable protein maintained at high levels for at least 6 days following its peak level of expression. Moreover, it is found predominantly in the soluble fraction of infected tissues. An immunocytochemical analysis of infected Chenopodium quinoa leaves reveals that p26 is exclusively associated with cytoplasmic inclusions in proximity to but distinct from aggregates of viral particles.
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PMID:Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein. 752 71

The 126 K protein of tobacco mosaic virus (TMV) is an NTP binding protein that has guanylyl transferase activity and is predicted to be an ATPase/helicase. In this paper we report the generation of monoclonal antibodies (Mabs) that react with specific amino acid sequences of the 126K protein. The Mabs were generated after immunizing mice with a partially purified preparation of the 126 K protein (native) obtained by centrifugal fractionation of the infected tissue extracts. The Mabs were assayed for specific reactivity by western blotting and by their reactivity with non-overlapping decapeptides corresponding to the entire amino acid sequence of the 126 K protein of TMV. A total of 11 Mabs reactive with specific peptides and three other Mabs that did not react with any peptide but reacted with the 126 K protein were identified.
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PMID:Monoclonal antibodies reactive with specific amino acid sequences of the 126 K protein of tobacco mosaic virus. 752 25

A complementary DNA encoding DNA-dependent ATPase Q1 possessing DNA helicase activity, which is the major DNA-dependent ATPase in human cell extracts, was cloned from a cDNA library of human KB cells. The predicted amino acid sequence has seven consecutive motifs conserved in the RNA and DNA helicase super family and DNA helicase Q1 belongs to DEXH helicase family. A homology search indicated that helicase Q1 had 47% homology in the seven conserved regions with Escherichia coli RecQ protein. Three RNA bands of 4.0, 3.3, and 2.2 kilobases were detected in HeLa cells by Northern blotting. Analysis of the genomic DNA indicated the presence of a homologous gene in mouse cells. The DNA helicase Q1 gene was localized on the short arm of human chromosome 12 at 12p12.
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PMID:Molecular cloning of cDNA encoding human DNA helicase Q1 which has homology to Escherichia coli Rec Q helicase and localization of the gene at chromosome 12p12. 752 36

By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single-stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate.
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PMID:Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454. 753 69

The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity. However, MBP-Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP-Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins.
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PMID:A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities. 753 73

The vaccinia virus A18R protein is a DNA-dependent ATPase that contains the canonical sequence motifs associated with the DEXH group of DNA and RNA helicases. Investigation of A18R protein function during infection indicated it functions in the early and late phases of vaccinia virus transcription. The A18R protein shares sequence similarity with the mammalian DNA helicase ERCC3. The ERCC3 protein has a dual function: it is a component of the transcription factor TFIIH and is an essential participant in the cellular nucleotide excision repair pathway. Here we present evidence that the A18R protein is a DNA helicase that unwinds duplex DNA in a 3'-to-5' direction. The A18R helicase was inactive on RNA-DNA and RNA-RNA hybrids. The A18R unwinding activity was most efficient on DNA substrates with lengths of 20 nucleotides or less, and its unwinding activity was not stimulated by the addition of Escherichia coli single-strand-binding protein (SSB), the bacteriophage T4 gene 32 SSB, or the vaccinia virus I3L protein, a putative SSB. We have used an electrophoretic gel mobility shift assay to show that the A18R protein forms a stable complex with single-stranded DNA, and to a lesser extent RNA, in a reaction that does not require ATP.
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PMID:Vaccinia virus gene A18R encodes an essential DNA helicase. 754 42


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