Search PubMed⌕ Search

Biomedical subjects

S Shuman

Publications and source records attributed to S Shuman.

At least 127 records · Page 7Linked to original sources
PubMed1996

Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin.

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K1 values of 10-25 microM for coumermycin and 350 microM for novobiocin. Spectroscopic and fluorescence measurements of drug binding t enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (KD = 27 +/- 5 microM) and two classes of binding sites for novobiocin, one tight site (KD1 = 20 +/- 5 microM) and several weak sites (KD2 = 513 +/- 125 microM; n = 4.9 +/- 0.7). Addition of a stoichiometric amount of DNA to a performed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.

Aminocoumarins↗
PubMed1996

Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.

Base Sequence↗
PubMed1996

Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1.

We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus. We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.

Chlorella↗
PubMed1996

Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription.

Temperature-sensitive mutations (ts10, ts18, and ts39) of the vaccinia virus RNA helicase nucleoside triphosphate phosphohydrolase II (NPH-II) result in the production of noninfectious progeny virions at the restrictive temperature. The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of Western blot (immunoblot) analysis indicate that these particles lack NPH-II, whereas other enzymatic components of the virus core are present. These components include the following: DNA-dependent RNA polymerase subunits rpo147, rpo132, rpo94, rpo35, rpo30, rpo22, and rpo18; early transcription initiation factor subunits A8 and D6; mRNA capping enzyme subunits D1 and D12; RNA cap 2'-O-methyltransferase; A18 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase. Although RNA polymerase is encapsidated by the mutant viruses, mRNA synthesis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporation into acid-insoluble material. Moreover, the transcripts synthesized by the mutant particles are longer than normal and remain virion associated. Transcription initiation by mutant virions occurs accurately at an endogenous genomic promoter, albeit at reduced levels (1 to 7%) compared with that of wild-type virions. In contrast, extracts of the mutant virions catalyze the wild-type level of transcription from an exogenous template containing an early promoter. We conclude that NPH-II is required for early mRNA synthesis uniquely in the context of the virus particle. Possible roles in transcription termination and RNA transport are discussed.

Acid Anhydride Hydrolases↗
PubMed1996

The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding.

Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent.

Adenosine Triphosphatases↗
PubMed1996

Mutational analysis of the vaccinia virus E3 protein defines amino acid residues involved in E3 binding to double-stranded RNA.

Alanine-substitution mutations were targeted to 14 amino acid residues within the double-stranded (ds) RNA binding motif (dsRBM) of the vaccinia virus E3 protein. Substitutions at six positions--Glu-124, Phe-135, Phe-148, Lys-167, Arg-168, and Lys-171--caused significant reductions in dsRNA binding. These six residues are conserved in the two dsRBMs for which structural information is available (Escherichia coli RNase III and Drosophila melanogaster staufen) and in many other members of the dsRBM protein family. Residues we show to be important for dsRNA binding by vaccinia virus E3 map to the same face of the dsRBM structure and are thus likely to compose part of the RNA binding site.

Amino Acid Sequence↗
PubMed1996

Vaccinia virus RNA helicase: nucleic acid specificity in duplex unwinding.

Vaccinia virus RNA helicase (NPH-II) catalyzes nucleoside triphosphate-dependent unwinding of duplex RNAs containing a single-stranded 3' RNA tail. In this study, we examine the structural features of the nucleic acid substrate that are important for helicase activity. Strand displacement was affected by the length of the 3' tail. Whereas NPH-II efficiently unwound double-stranded RNA substrates with 19- or 11-nucleotide (nt) 3' tails, shortening the 3' tail to 4 nt reduced unwinding by an order of magnitude. Processivity of the helicase was inferred from its ability to unwind a tailed RNA substrate containing a 96-bp duplex region. NPH-II exhibited profound asymmetry in displacing hybrid duplexes composed of DNA and RNA strands. A 34-bp RNA-DNA hybrid with a 19-nt 3' RNA tail was unwound catalytically, whereas a 34-bp DNA-RNA hybrid containing a 19-nt 3' DNA tail was 2 orders of magnitude less effective as a helicase substrate. NPH-II was incapable of displacing a 34-bp double-stranded DNA substrate of identical sequence. 3'-Tailed DNA molecules with 24- or 19-bp duplex regions were also inert as helicase substrates. On the basis of current models for RNA-DNA hybrid structures, we suggest the following explanation for these findings. (i) Unwinding of duplex nucleic acids by NPH-II is optimal when the polynucleotide strand of the duplex along which the enzyme translocates has adopted an A-form secondary structure, and (ii) a B-form secondary structure impedes protein translocation through DNA duplexes.

Base Sequence↗
PubMed1996

Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme.

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities. The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions. Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant enzymes were less active than wild-type D1(1-545) in the capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage. Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of the vaccinia virus capping enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting.

Acid Anhydride Hydrolases↗
PubMed1996

Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

Alanine↗
PubMed1996

Conditional inactivation of mRNA capping enzyme affects yeast pre-mRNA splicing in vivo.

Acquisition of the 5'cap is the earliest modification event during eukaryotic mRNA synthesis. The cap is thought to facilitate later processing steps, such as pre-mRNA splicing. If this is so, then a defect in cap synthesis should impact on splicing in vivo. We tested this hypothesis by examining the consequences of conditional inactivation of the Saccharomyces cerevisiae CEG1 gene, which encodes mRNA guanylyltransferase (capping enzyme). Two different ceg1-ts mutants, Y66A and C354Y, displayed a pre-mRNA processing (prp) defect, characterized by an increase in the amount of unspliced pre-mRNA after shift to nonpermissive temperature and a decrease in the amount of mature mRNA. The guanylyltransferase activities of the Y66A and C354Y proteins were thermolabile, suggesting that defective capping in vivo was contributory to the prp phenotype. Although these results provide the first genetic link between capping and splicing in vivo, we were unable to demonstrate a role for either the cap or the capping enzyme during yeast pre-mRNA splicing in vitro.

Alleles↗
PubMed1996

Multicopy suppressors of temperature-sensitive mutations of yeast mRNA capping enzyme.

We have isolated three Saccharomyces cerevisiae genes-CES1, CES2, and CES3-- that, when present in high copy, suppress the ts growth defect caused by mutations in the CEG1 gene encoding mRNA guanylyltransferase (capping enzyme). Molecular characterization of the capping enzyme suppressor genes reveals the following. CES2 is identical to ESP1, a gene required for proper nuclear division. We show by deletion analysis that the 1573-amino acid ESP1 polypeptide is composed of distinct functional domains. The C-terminal portion of ESP1 is essential for cell growth, but dispensable for CES2 activity. The N-terminal half of ESP1, which is sufficient for CES2 function, displays local sequence similarity to the small subunit of the vaccinia virus RNA capping enzyme. This suggests a basis for suppression by physical or functional interaction between the CES2 domain of ESP1 and the yeast guanylyltransferase. CES1 encodes a novel hydrophilic 915-amino acid protein. The amino acid sequence of CES1 is uninformative, except for its extensive similarity to another yeast gene product of unknown function. The CES1 homologue (designated CES4) is also a multicopy suppressor of capping enzyme ts mutations. Neither CES1 nor CES4 is essential for cell growth, and a double deletion mutant is viable. CES3 corresponds to BUD5, which encodes a putative guanine nucleotide exchange factor. We hypothesize that CES1, CES4, and BUD5 may impact on RNA transactions downstream of cap synthesis that are cap dependent in vivo.

Amino Acid Sequence↗
PubMed1995

Vaccinia virus DNA ligase: specificity, fidelity, and inhibition.

Vaccinia DNA ligase and mammalian DNA ligases II and III comprise a distinct subgroup of structurally homologous enzymes within the eukaryotic DNA ligase family. The specificity and fidelity of the viral enzyme were investigated using purified recombinant ligase and synthetic duplex DNA substrates containing a single strand discontinuity. Vaccinia ligase catalyzed efficient strand joining on nicked DNAs in the presence of magnesium and ATP (Km = 95 microM). dATP, ITP, AMPPCP, 3'dATP, and ATP alpha S could not substitute for ATP; of these, 3'dATP and ATP alpha S were inhibitors of ligation. The vaccinia enzyme was unable to seal strands across a 1 nt (nucleotide) or 2 nt gap. Ligase action at a 1 nt gap resulted in accumulation of high levels of the normally undetectable DNA-adenylate reaction intermediate. In contrast, no DNA-adenylate was formed at a 2 nt gap. A native gel mobility shift assay showed that vaccinia DNA ligase was capable of discriminating between nicked and gapped DNAs at the substrate binding step. The ligase was fairly tolerant of mismatches at a nick involving the 5' phosphate donor terminus but was inhibited strongly by mismatches at the 3' OH acceptor terminus, especially by purine.-purine mispairs. These findings underscore the importance of a proper 3' OH terminus in substrate recognition and reaction chemistry but also raise the possibility that ligase may generate mutations during DNA repair by sealing DNA molecules with mispaired ends. Ligase was inhibited by several DNA binding drugs, including, in order of decreasing potency, distamycin, ethidium bromide, and actinomycin. Strand joining by purified ligase was not affected by etoposide, a drug that inhibits vaccinia virus replication in vivo and which depends on the presence of vaccinia ligase for its antiviral action.

Adenosine Triphosphate↗
PubMed1995

Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis.

DNA ligation entails AMP transfer from ATP to the 5' end of DNA to form a DNA-adenylate structure, A(5')pp(5')N. A similar reaction involving GMP transfer occurs during 5' capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase-AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.

Amino Acid Sequence↗
PubMed1995

Proteolytic footprinting of vaccinia topoisomerase bound to DNA.

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent protein.DNA complex at sites containing a conserved sequence element 5'-CCCTT decreases. The structure of the enzyme in the free and DNA-bound states was probed by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease-resistant amino- and carboxyl-terminal structural domains flanking a protease-sensitive "hinge." The hinge region, located between residues 135 and 142, is defined by accessibility to three different proteases. The amino-terminal region is punctuated by a trypsin-sensitive "bridge" at Arg-80, suggesting at least a tripartite domain structure overall. A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state. The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex. Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively. Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation. The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase.

Amino Acid Sequence↗
PubMed1995

RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases.

mRNA capping entails GMP transfer from GTP to a 5' diphosphate RNA end to form the structure G(5')ppp(5')N. A similar reaction involving AMP transfer to the 5' monophosphate end of DNA or RNA occurs during strand joining by polynucleotide ligases. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. Sequence conservation among capping enzymes and ATP-dependent ligases in the vicinity of the active site lysine (KxDG) and at five other co-linear motifs suggests a common structural basis for covalent catalysis. Mutational studies support this view. We propose that the cellular and DNA virus capping enzymes and ATP-dependent ligases constitute a protein superfamily evolved from a common ancestral enzyme. Within this superfamily, the cellular capping enzymes display more extensive similarity to the ligases than they do to the poxvirus capping enzymes. Recent studies suggest that eukaryotic RNA viruses have evolved alternative pathways of cap metabolism catalysed by structurally unrelated enzymes that nonetheless employ a phosphoramidate intermediate. Comparative analysis of these enzymes, particularly at the structural level, should illuminate the shared reaction mechanism while clarifying the basis for nucleotide specificity and end recognition. The capping enzymes merit close attention as potential targets for antiviral therapy.

Amino Acid Sequence↗
PubMed1995

Adenosine N1-oxide inhibits vaccinia virus replication by blocking translation of viral early mRNAs.

Adenosine N1-oxide (ANO) is a potent and highly selective inhibitor of vaccinia virus replication. We examined the impact of ANO on vaccinia virus macromolecular synthesis during synchronous infection of BSC40 cells. Viral DNA replication and viral late protein synthesis were blocked completely by ANO, effects that were attributable to a defect in the expression of viral early genes. Vaccinia virus early proteins were not synthesized in the presence of ANO, even though vaccinia virus early mRNAs were produced. Cellular protein synthesis was unaffected by ANO, and virus infection in the presence of the drug did not elicit the normal shutoff of host protein synthesis. Adenosine N1-oxide triphosphate (ANO-TP), the predominant metabolite of the drug in vivo, could substitute for ATP in RNA synthesis by purified vaccinia virus RNA polymerase. ANO-TP could support early transcription by purified virions if dATP was provided as an energy source. ANO-TP did not inhibit early transcription in the presence of ATP. These findings suggest a novel antiviral mechanism whereby incorporation of a modified nucleotide into viral mRNAs might selectively block viral gene expression at the level of translation. We believe that ANO merits consideration as an antipoxvirus drug for topical treatment of molluscum contagiosum in humans.

Adenosine↗
PubMed1995

Vaccinia virus morphogenesis is blocked by temperature-sensitive mutations in the F10 gene, which encodes protein kinase 2.

Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts28, ts54, ts61, and ts15) composing a single complementation group have been mapped by marker rescue to the F10 open reading frame located within the genomic HindIII F DNA fragment. Sequencing of the F10 gene from wild-type and mutant viruses revealed single-amino-acid substitutions in the F10 polypeptide responsible for thermolabile growth. Although the ts mutants displayed normal patterns of viral protein synthesis, electron microscopy revealed a profound morphogenetic defect at the nonpermissive temperature (40 degrees C). Virion assembly was arrested at an early stage, with scant formation of membrane crescents and no progression to normal spherical immature particles. The F10 gene encodes a 52-kDa serine/threonine protein kinase (S. Lin and S. S. Broyles, Proc. Natl. Acad. Sci. USA 91:7653-7657, 1994). We expressed a His-tagged version of the wild-type, ts54, and ts61 F10 polypeptides in bacteria and purified these proteins by sequential nickel affinity and phosphocellulose chromatography steps. The wild-type F10 protein kinase activity was characterized in detail by using casein as a phosphate acceptor. Whereas the wild-type and ts61 kinases displayed similar thermal inactivation profiles, the ts54 kinase was thermosensitive in vitro. These findings suggest that protein phosphorylation plays an essential role at an early stage of virion assembly.

Amino Acid Sequence↗