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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)

Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.
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PMID:Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover. 171 28

Two DNA helicases from calf thymus nuclei have been purified and characterized. The two proteins, designated as nuclear DNA helicase I and II, were copurified on Bio-Rex 70, DEAE-Sepharose, phosphocellulose and subsequently separated from each other on a heparin-Sepharose column. Final purification of DNA helicase I was achieved on single-stranded DNA-cellulose and that of DNA helicase II on ATP-agarose. On denaturing polyacrylamide gels, nuclear DNA helicase I displayed two polypeptide bands of 170 and 200 kDa; nuclear DNA helicase II also consisted of two bands, in this case of 100 and 130 kDa. Both enzymes catalyzed the unwinding of a DNA primer from a single-stranded DNA template but had different nucleotide requirements for their energy supply. While nuclear DNA helicase I preferred to hydrolyze ATP or dATP to support its unwinding activity, nuclear DNA helicase II could utilize all four rNTPs or dNTPs, though ATP or dATP were still preferred to other nucleotides. ADP, AMP, or adenosine 5'-O-(thiotriphosphate) could not be used by either enzyme in the unwinding reactions. A divalent cation was essential for activity of both enzymes. Nuclear DNA helicase I required 3-5 mM Mg2+ or 1 mM Mn2+ for optimal unwinding. In contrast, nuclear DNA helicase II displayed high activity even at very low concentrations of Mg2+. Nuclear DNA helicase I was stimulated by NaCl, KCl, or potassium acetate up to concentrations of 150 mM; in contrast, nuclear DNA helicase II was strongly inhibited at salt concentrations over 75 mM. Both DNA helicases had an associated ATPase activity. However, while the ATPase activity of nuclear DNA helicase I was stimulated only in presence of single-stranded DNA, the ATPase activity of the nuclear DNA helicase II was stimulated by single-stranded DNA and, even more efficiently, by RNA. Finally, the translocation of both DNA helicases had a polarity from 3' to 5' with respect to the single-stranded DNA template to which the enzymes were bound. A comparison of these DNA helicases with the other reported mammalian DNA helicases will be given. The significance of the association of the two DNA helicases during the process of the purification will be discussed.
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PMID:Purification and characterization of two DNA helicases from calf thymus nuclei. 171 63

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. The RAD3 protein exhibits a remarkable degree of sequence homology to the human excision repair protein ERCC2. The RAD3 protein is a single-stranded DNA-dependent ATPase and a DNA helicase capable of denaturing long regions of duplex DNA. Here, we demonstrate that RAD3 also possesses a potent DNA.RNA helicase activity similar in efficiency to its DNA helicase activity. The rad3 Arg-48 mutant protein, which binds but does not hydrolyze ATP, lacks the DNA.RNA unwinding activity, indicating a dependence on ATP hydrolysis. RAD3 does not show any RNA-dependent NTPase activity and, as expected, does not unwind duplex RNA. This observation suggests that RAD3 translocates on DNA in unwinding DNA.RNA duplexes. That the rad3 Arg-48 mutation inactivates the DNA and DNA.RNA helicase activities and confers a substantial reduction in the incision of UV-damaged DNA suggests a role for these activities in incision. We discuss how RAD3 helicase activities could function in tracking of DNA in search of damage sites and effect enhanced excision repair of actively transcribed genes.
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PMID:DNA.RNA helicase activity of RAD3 protein of Saccharomyces cerevisiae. 171 38

A recombinant herpes simplex 1 origin binding protein, the product of the herpes UL9 gene, has been overexpressed in mammalian cells and purified to near homogeneity. The origin binding protein shows DNA-dependent nucleoside 5'-triphosphatase and DNA helicase activities in addition to its origin binding activity. The ability to hydrolyze nucleoside 5'-triphosphates is influenced strongly by the structure and sequence of the DNA cofactor. The properties of the recombinant origin binding protein are identical to those of the protein synthesized in herpes simplex 1-infected mammalian cells.
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PMID:The herpes simplex virus 1 origin binding protein: a DNA helicase. 184 32

Herpes simplex virus 1 encodes a helicase-primase that is composed of the products of the UL5, UL8, and UL52 genes. A stable subassembly consisting of only the UL5 and UL52 gene products has been purified to near homogeneity from insect cells doubly infected with baculovirus recombinant for these two genes. The purified subassembly has the DNA-dependent ATPase, DNA-dependent GTPase, DNA helicase, and DNA primase activities that are characteristic of the three-subunit holoenzyme. The purified UL8 gene product, although required for viral DNA replication, neither exhibits these enzymatic activities nor stably associates with either the UL5 or the UL52 gene product.
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PMID:Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products. 184 9

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix-localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single-stranded (ss) DNA-dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5' to 3' direction with respect to the ss DNA on which it binds first.
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PMID:PIF1: a DNA helicase in yeast mitochondria. 184 81

A DNA helicase activity was detected in extracts of purified chloroplasts from the SB-1 cell line of Glycine max and partially purified by column chromatography on DEAE cellulose, phosphocellulose, and single-stranded DNA cellulose. The chloroplast helicase has a DNA-dependent ATPase activity, and its strand displacement activity is strictly dependent upon the presence of a nucleoside triphosphate and Mg2+ or Mn2+. Strand displacement activity does not require a free unannealed single-strand or replication fork-like structure.
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PMID:Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max. 196 89

We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound.
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PMID:DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme. 197 96

A consensus sequence in parvovirus nonstructural protein NS1 has been predicted to be an ATP-binding domain associated with an ATPase and a DNA helicase activity. To investigate the function of NS1 in viral gene expression, a site-directed mutagenesis converting NS1 lysine 405 to serine in parvovirus H-1 was carried out by the polymerase chain reaction. As shown previously, a parvovirus genome containing a deleted NS1 gene was excised from a bacterial plasmid and replicated when a wild-type NS1 gene was provided in trans but failed to be excised and replicate when the mutant NS1 gene was supplied. Interestingly, the serine 405 mutation totally lost the activity of trans activation on the virus late promoter (P38) in a chloramphenicol acetyltransferase (CAT) assay and it lost evidence for cytotoxicity in two tumor cell lines (HeLa Gey and NB324K). The serine 405 NS1 protein was translocated normally to the nucleus. These results suggest that the NS1 lysine 405 of H-1 in its putative purine nucleotide-binding site is essential for viral DNA replication and that this domain may be involved in the regulation of the P38 promoter by an unknown mechanism. The loss of NS1 cytotoxicity on tumor cells suggests that NS1 expression is the major cause of cell killing by parvoviruses, which may facilitate further study of the mechanism of oncosuppression by parvoviruses.
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PMID:Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity. 214 94

Biochemical evidence is presented that confirms exonuclease V of Escherichia coli consists of three distinct subunits encoded by the recB, recC, and recD genes. The recD gene encodes a Mr 60,000 polypeptide and physically maps 3' to the recB structural gene. The role of the recD subunit in exonuclease V function has been examined by comparing the catalytic activities of the purified RecBCD enzyme with the RecBC enzyme. The RecBC enzyme retains significant levels of DNA-dependent ATPase activity and DNA helicase activity. Endonucleolytic activity on single-stranded covalently closed DNA becomes ATP-dependent. Exonucleolytic activity on either single- and double-stranded DNA was not detected. Taken together with the phenotypic properties of recD null mutants, it appears that the exonucleolytic activities of the RecBCD enzyme are not required for genetic recombination and the repair of either UV-induced photoproducts or mitomycin C-generated DNA cross-links, but are essential for the repair of methyl methanesulfonate-induced methylation.
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PMID:Biochemical and physical characterization of exonuclease V from Escherichia coli. Comparison of the catalytic activities of the RecBC and RecBCD enzymes. 215 79


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