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)

A recombinant baculovirus overexpressing the herpes simplex virus type 1 (HSV-1) origin binding protein, encoded by the UL9 gene, was constructed. The purified recombinant protein has DNA-dependent nucleoside triphosphatase activity similar to the enzyme isolated from mammalian cells. Optimal nucleoside triphosphatase activity requires low salt (< 50 mM), 2-3 mM Mg2+, alkaline pH (8.3-9.5), high temperature (45 degrees C), and a single-stranded DNA coeffector containing minimal secondary structure. Enzymatic activity is subject to product inhibition, and there appears to be a single nucleotide binding site. The minimal length of single-stranded DNA that elicits enzymatic activity is 14 nucleotides, and activity increases as the length is increased. Saturation for various single-stranded DNA coeffectors is about 10 microM in nucleotide, but the maximum velocity is reduced 2-3-fold for coeffectors containing secondary structure. The HSV-1-encoded single-stranded DNA-binding protein ICP8 specifically stimulates the DNA-dependent nucleoside triphosphatase activity. The kinetics of nucleoside triphosphate hydrolysis exhibit a substantial lag period which can be shortened, but not eliminated, by reduced secondary structure in the DNA coeffector or by increased temperature.
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PMID:The herpes simplex virus type I origin binding protein. DNA-dependent nucleoside triphosphatase activity. 838 Apr 7

Herpes simplex virus type 1 encodes a helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins purified from insect cells also displays ATPase, helicase, and primase activities. UL5 contains six motifs conserved in superfamily I of known and/or putative helicase proteins. Consistent with the ability to hydrolyze ATP, motifs I and II resemble a nucleotide binding site. Although the role of the other four motifs is not known, single amino acid substitutions created in conserved residues in all six motifs abolish the ability of UL5 to support viral DNA replication in vivo (Zhu, L., and Weller, S. K. (1992) J. Virol. 66, 469-479). In one such mutation, a highly conserved glycine in motif V (Gly815) is replaced with an alanine. Although the UL5(G815A) protein does not support viral DNA replication in vivo, the purified UL5(G815A).52 subcomplex retains primase and helicase activities and supports strand displacement DNA synthesis on a preformed replication fork in the presence of the other HSV-1 replication proteins. The major difference between the wild-type and variant protein is that the UL5(G815A).52 subcomplex displays an increased Km for single-stranded DNA and decreased Kcat for single-stranded DNA-dependent ATPase activity. Several hypotheses for the role of motif V in the function of the UL5 helicase in HSV-1 DNA replication are considered. This is the first report of a biochemical analysis of a motif V variant in any member of helicase superfamily I.
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PMID:Replacement of gly815 in helicase motif V alters the single-stranded DNA-dependent ATPase activity of the herpes simplex virus type 1 helicase-primase. 866 72

The herpes simplex virus type-1 DNA helicase-primase is a heterotrimer encoded by the UL5, UL8, and UL52 genes. The core enzyme, specified by the UL5 and UL52 genes, retains DNA helicase, DNA-dependent nucleoside triphosphatase, and primase activities. The UL8 subunit has previously been implicated in increasing primer stability and in stimulating primer synthesis by the core enzyme. To further characterize the function of the UL8 subunit, we have examined its effect on the activities of the UL5/52 core enzyme using DNA templates covered by the herpes simplex virus type-1 single-strand DNA-binding protein ICP8. We found that while ICP8 stimulated the DNA helicase activity of the UL5/52 proteins up to 3-fold, maximum stimulation by ICP8 required the presence of UL8 protein. Moreover, UL8 protein was required to reverse the inhibitory effect of ICP8 on the DNA-dependent ATPase and primase activities of the UL5/52 proteins. These observations were specific for ICP8 since the heterologous Escherichia coli single-strand DNA-binding protein could not substitute for ICP8. These data suggest that UL8 protein mediates an interaction between the UL5/52 core enzyme and ICP8 that optimizes the utilization of ICP8-covered DNA templates during DNA replication.
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PMID:The UL8 subunit of the herpes simplex virus type-1 DNA helicase-primase optimizes utilization of DNA templates covered by the homologous single-strand DNA-binding protein ICP8. 870 53

The UL9 protein of herpes simplex virus type 1 binds to specific sequences within the viral origins of DNA replication and also functions as a DNA helicase. The C-terminal 317 amino acids of the 851 residue protein specify sequence-specific binding to the viral origins and the N-terminal 400 contain several motifs characteristic of many DNA and RNA helicases. To investigate whether the origin-binding domain is required for helicase function we have expressed a truncated version comprising amino acids 1-535 of UL9 using a recombinant baculovirus. Extracts were prepared from cells infected with the recombinant virus and chromatographed over ATP-agarose. DNA helicase, DNA-dependent ATPase and a novel single-stranded DNA-binding activity were present in fractions containing the truncated UL9 protein but not in corresponding gradient fractions from a control virus infection. These results indicate that the DNA helicase function of UL9 does not require the presence of the origin-binding domain and suggest that an interaction between the N-terminal domain and distorted or partially single-stranded regions of DNA may play a role in unwinding the origin region.
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PMID:The origin-binding domain of the herpes simplex virus type 1 UL9 protein is not required for DNA helicase activity. 884 19

The UL5, UL8, and UL52 genes of herpes simplex virus type 1 encode a multisubunit assembly that possesses primase, DNA helicase, and DNA-dependent nucleoside triphosphatase activities. A subassembly consisting of the UL5 and UL52 gene products retains these activities. The nucleoside triphosphatase activity of the UL5/UL52 subassembly is strongly stimulated by both homo- and heteropolymeric single-stranded DNA. Double-stranded DNA has little ability to stimulate the ATPase activity. The subassembly binds both double and single-stranded DNA. Nucleotides are not required for DNA-binding. The minimum length of single-stranded DNA that is bound and that stimulates enzymatic activity is about 12 nucleotides. The kinetic parameters of the ATPase activity of the subassembly are affected by the length of the oligonucleotide coeffector. The Km decreases as the coeffector length is increased up to a length of about 20 nucleotides and then remains independent of coeffector length. The first order rate constant for ATPase activity exhibits a quasihyperbolic dependence on the length of the DNA coeffector and is maximal for coeffectors of 20 nucleotides and longer.
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PMID:Interactions of a subassembly of the herpes simplex virus type 1 helicase-primase with DNA. 901 84

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase composed of the products of the UL5, UL52, and UL8 genes. UL5 possesses six motifs conserved among superfamily 1 of helicase proteins. Substitutions of conserved residues in each motif abolishes DNA replication in vivo (Zhu, L., and Weller, S. K. (1992) J. Virol. 66, 469-479). Purified UL5.52 harboring a Gly to Ala change in motif V retains primase and helicase activities in vitro but exhibits a higher KM for single-stranded DNA and lower DNA-dependent ATPase activity (Graves-Woodward, K. L., and Weller, S. K. (1996) J. Biol. Chem. 272, 13629-13635). We have purified and characterized six other subcomplexes with residue changes in the UL5 helicase motifs. Each variant subcomplex displays at least wild type or greater levels of primase and DNA binding activities, but all are defective in helicase activity. Mutations in motifs I and II exhibit profound decreases in DNA-dependent ATPase activity. Mutations in motifs III-VI decrease DNA-dependent ATPase activity 3-6-fold. Since mutations in motifs III, IV, V, and VI do not eliminate ATP hydrolysis or DNA binding, we propose that they may be involved in the coupling of these two activities to the process of DNA unwinding. This analysis represents the first comprehensive structure-function analysis of the conserved motifs in helicase superfamily 1.
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PMID:Biochemical analyses of mutations in the HSV-1 helicase-primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding. 902 Jan 91

A new continuous coupled uv-spectrophotometric assay is described for two phosphate-releasing enzymes, aspartate transcarbamylase and ATPase of herpes simplex virus (HSV). Phosphate release is coupled to the phosphorolysis of the nucleoside analog 7-methylinosine (m7Ino) catalyzed by purine nucleoside phosphorylase. When this reaction is monitored at 291 nm, the coupled assay can readily detect 10 nmol Pi released/min. Our method offers advantages over a recently reported continuous assay devised for measuring aspartate transcarbamylase activity using the nucleoside analog methylthioguanosine (MESG) as the linking substrate. In contrast to MESG, m7Ino is easily and inexpensively synthesized and is also commercially available. The spectrophotometric signal at 291 nm, produced by the difference in the extinction coefficients between nucleoside substrate and the base product, is significant over a much wider pH range than the signal difference between MESG and its phosphorolysis product at 360 nm. Saturation curves for aspartate and carbamyl phosphate and pH rate profiles have been reproduced using the purine nucleoside phosphorylase/m7Ino coupled assay. Initial velocity patterns constructed over micromolar to millimolar concentrations of aspartate and carbamyl phosphate yielded four kinetic parameters simultaneously. To further illustrate the application of this coupled assay, kinetic parameters were determined for the DNA-dependent ATPase reaction of HSV helicase-primase.
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PMID:A continuous spectrophotometric assay for aspartate transcarbamylase and ATPases. 905 87

The herpes simplex virus type 1 (HSV) single-stranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.
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PMID:A functional interaction of ICP8, the herpes simplex virus single-stranded DNA-binding protein, and the helicase-primase complex that is dependent on the presence of the UL8 subunit. 912 59

The Herpes simplex virus type 1 primosome consists of three subunits that are the products of the UL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a single-stranded DNA loading site fully coated with the virus-encoded single strand DNA binding protein, ICP8. The Escherichia coli single strand DNA binding protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to single-stranded DNA but not to ICP8-coated DNA.
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PMID:The UL8 subunit of the heterotrimeric herpes simplex virus type 1 helicase-primase is required for the unwinding of single strand DNA-binding protein (ICP8)-coated DNA substrates. 927 36

R13-1 is an intertypic recombinant virus in which the left-hand 18% of the herpes simplex virus type 1 (HSV-1) genome is replaced by homologous sequences from HSV-2. R13-1 is nonneurovirulent and defective in DNA replication in neurons. The defect was localized to the UL5 open reading frame by using marker rescue analysis (D. C. Bloom and J. G. Stevens, J. Virol. 68:3761-3772, 1994). To provide conclusive evidence that UL5 is the only HSV-2 gene involved in the restricted replication phenotype of R13-1, we have characterized the phenotype of a recombinant virus (IB1) in which only the UL5 gene of HSV-1 was replaced by HSV-2 UL5. Data from 50% lethal dose determinations and the in vivo yields of virus suggested that IB1 has the same phenotypic characteristics as R13-1. UL5 is the helicase component of a complex with helicase and primase activities. All three subunits of this complex (UL5, UL8, and UL52) are required for viral DNA replication in all cell types. The intertypic complex HSV-2 UL5-HSV-1 UL8-HSV-1 UL52 was purified and biochemically characterized. The primase activity of the intertypic complex was 10-fold lower than that of HSV-1 UL5-HSV-1 UL8-HSV-1 UL52. The ATPase activity was comparable to that of the HSV-1 enzyme complex, and although the helicase activity was threefold lower, this did not interfere with the synthesis of leading strands by the HSV polymerase. One explanation for these findings is that the interactions between the subunits of the helicase-primase intertypic complex that are important for the full function of each subunit are inappropriate or weak.
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PMID:An intertypic herpes simplex virus helicase-primase complex associated with a defect in neurovirulence has reduced primase activity. 944 19


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