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S Shuman

Publications and source records attributed to S Shuman.

At least 163 records · Page 9Linked to original sources
PubMed1993

Site-specific interaction of vaccinia virus topoisomerase I with base and sugar moieties in duplex DNA.

Vaccinia DNA topoisomerase specifically binds and forms a covalent adduct at DNA sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. The molecular interactions that contribute to recognition of the CCCTT motif in a synthetic DNA substrate have been examined using modification interference, modification protection, and analog substitution techniques. We report that topoisomerase makes contact with guanine nucleotide bases of the pentamer motif complementary strand (3'GGGAA) within the major groove of the DNA helix and that alteration of the binding surface by chemical modification is deleterious to the interaction. Additional contacts are made with guanine residues located outside the pentamer element. The enzyme is unable to form a covalent adduct with synthetic RNA substrates. Analysis of the cleavage of DNA duplexes containing 2'OMe sugars suggests that the inability of the vaccinia topoisomerase to cleave either an RNA duplex or an RNA:DNA hybrid can be accounted for by the interfering effects of a 2' sugar substituent at two or more sites within the pentamer. Interaction with the sugar at the +2T nucleotide appears to be the most critical, as judged by the effects of single sugar substitutions.

Base Sequence↗
PubMed1993

Vaccinia virus RNA helicase. Directionality and substrate specificity.

Vaccinia virus RNA helicase (NPH-II) catalyzes unidirectional unwinding of 3'-tailed duplex RNAs in the presence of a divalent cation and any one of the eight common nucleoside triphosphates (NTP). The helicase binds stably to the tailed RNA in the absence of any cofactor; strand displacement by the bound protein is coupled to NTP hydrolysis. Although the helicase is capable of binding to tailed duplex DNA as well as to tailed RNA, the enzyme is unable to unwind duplex DNA. It is suggested that NTP hydrolysis by RNA-bound NPH-II drives processive translocation of the protein in a 3' to 5' direction along the RNA strand, whereas energy utilization by DNA-bound enzyme leads to dissociation without extensive protein movement. A role for the RNA helicase in vaccinia mRNA synthesis is proposed.

Adenosine Triphosphatases↗
PubMed1993

Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases.

Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.

Amino Acid Sequence↗
PubMed1993

Nascent RNA cleavage by purified ternary complexes of vaccinia RNA polymerase.

Ternary complexes of vaccinia virus RNA polymerase containing 3'-OMeGMP-arrested transcripts were purified by native gel electrophoresis. These complexes resumed elongation in situ when gel slices were incubated with magnesium and NTPs. Elongation occurred in the absence of pyrophosphate, suggesting that the blocking 3'-OMeGMP residue was removed via a novel pathway. We show that purified elongation complexes contain an intrinsic nuclease activity that shortens nascent RNA from the 3'-end. RNA cleavage was absolutely dependent on a divalent cation and was stimulated by CTP. The initial 5' cleavage product remained associated with the ternary complex and could be elongated in the presence of NTPs. Multiple stepwise cleavages generated progressively shorter chains. Purified ternary complexes containing 3'-OH-terminated RNAs also displayed nuclease activity. Involvement of the vaccinia RNA polymerase subunit rpo30 in the transcript-shortening reaction is suggested based on sequence similarity of rpo30 to mammalian protein SII (TFIIS), an extrinsic transcription factor required for nascent RNA cleavage by RNA polymerase II (Reines, D. (1991) J. Biol. Chem. 267, 3795-3800).

Base Sequence↗
PubMed1993

Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component.

The ts16 mutation of vaccinia virus WR (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) has been mapped by marker rescue to the I7L open reading frame located within the genomic HindIII I DNA fragment. The I7 gene encodes a 423-amino-acid polypeptide. Thermolabile growth was attributed to an amino acid substitution, Pro-344-->Leu, in the predicted I7 protein. A normal temporal pattern of viral protein synthesis was elicited in cells infected with ts16 at the nonpermissive temperature (40 degrees C). Electron microscopy revealed a defect in virion assembly at 40 degrees C. Morphogenesis was arrested at a stage subsequent to formation of spherical immature particles. Western immunoblot analysis with antiserum directed against the I7 polypeptide demonstrated an immunoreactive 47-kDa polypeptide accumulating during the late phase of synchronous vaccinia virus infection. Immunoblotting of extracts of wild-type virions showed that the I7 protein is encapsidated within the virus core. The I7 polypeptide displays amino acid sequence similarity to the type II DNA topoisomerase of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1992

Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases.

Three distinct nucleic acid-dependent ATPases are packaged within infectious vaccinia virus particles; one of these enzymes (nucleoside triphosphate phosphohydrolase II or NPH-II) is activated by single-stranded RNA. Purified NPH-II is now shown to be an NTP-dependent RNA helicase. RNA unwinding requires a divalent cation and any one of the eight common ribo- or deoxyribonucleoside triphosphates. The enzyme acts catalytically to displace an estimated 10-fold molar excess of duplex RNA under in vitro reaction conditions. NPH-II binds to single-stranded RNA. Turnover of the bound enzyme is stimulated by and coupled to hydrolysis of NTP. Photocrosslinking of radiolabeled RNA to NPH-II results in label transfer to a single 73-kDa polypeptide. The sedimentation properties of the helicase are consistent with NPH-II being a monomer of this protein. Immunoblotting experiments identify NPH-II as the product of the vaccinia virus I8 gene. The I8-encoded protein displays extensive sequence similarity to members of the DE-H family of RNA-dependent NTPases. Mutations in the NPH-II gene [Fathi, Z. & Condit, R.C. (1991) Virology 181, 258-272] define the vaccinia helicase as essential for virus replication in vivo. Encapsidation of NPH-II in the virus particle suggests a role for the enzyme in synthesis of early messenger RNAs by the virion-associated transcription machinery.

Amino Acid Sequence↗
PubMed1992

Structural analysis of ternary complexes of vaccinia RNA polymerase.

The structure of the elongation complex of vaccinia RNA polymerase halted at discrete template positions was examined by DNase I footprinting. The leading edge of the footprint bore a constant relationship to the catalytic template position, being 22-24 nucleotides (nt) in advance on the nontemplate strand and 17 nt on the template strand. DNase hypersensitivity of the nontemplate strand at the leading edge suggested that the DNA might be distorted as it entered the polymerase molecule. The region of DNA unwinding at the transcription bubble extended at least 12 nt 5' from the catalytic center, as indicated by the reactivity of adenosine residues to diethylpyrocarbonate. Cu-phenanthroline-hypersensitive sites located 13 nt 5' and 4 nt 3' of the growing point appeared to demarcate the margins of the bubble. Strand asymmetry of chemical modification within the bubble was consistent with an RNA-DNA hybrid of no more than 10 base pairs.

Base Sequence↗
PubMed1992

Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase I.

The ability of a eukaryotic DNA topoisomerase I to catalyze DNA rearrangements was examined in vitro using defined substrates and purified enzyme. Site-specific DNA strand cleavage by vaccinia topoisomerase I across from a nick generated double-strand breaks that could be religated to a heterologous blunt-ended duplex DNA regardless of the sequence of the acceptor molecule. Topoisomerase bound covalently at internal positions could religate the bound strand to an incoming acceptor provided that DNA molecule had sequence homology to the region 3' of the scissile bond. These end-joining reactions suggest two potential modes of topoisomerase-mediated recombination that differ in their requirements for DNA homology.

Base Sequence↗
PubMed1992

Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme.

RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7-)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of M(r) 95,000 and 31,000. Although the RNA triphosphatase and RNA guanylyltransferase domains have been localized to a M(r) 59,000 fragment of the capping enzyme large subunit, the location of the methyltransferase domain within the protein and the catalytic role of individual subunits in methyl group transfer remain unclear. In the present work, through the study of methyltransferase activity of truncated forms of capping enzyme translated in vitro in a rabbit reticulocyte lysate, we have localized the methyltransferase domain to a complex consisting of the small subunit and the carboxyl-terminal portion of the large subunit. The M(r) 31,000 subunit translated alone was not sufficient for methyltransferase activity. This requirement for both subunits may explain the tight physical association of the two polypeptides in vivo. We have recreated the association of the large and small enzyme subunits in vitro through the translation of synthetic mRNAs encoding the two polypeptides. Study of the ability of deleted versions of the large subunit to bind the small subunit, as detected by co-immunoprecipitation, defined a 347-amino acid carboxyl-terminal region of the large subunit that was sufficient for heterodimerization. Colocalization within the large subunit of the methyltransferase and subunit association domains suggests that dimerization of the subunits may be required for methyltransferase activity.

Animals↗
PubMed1992

Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I.

Analysis of vaccinia topoisomerase mutants that are impaired in DNA relaxation has allowed the identification of amino acid residues required for the transesterification step of catalysis. Missense mutations of wild-type residues Gly-132----Asp and Arg-223----Gln rendered the protein inert in formation of the covalent enzyme-DNA complex and hence completely inactive in DNA relaxation. Mutations of Thr-147----Ile and Gly-132----Ser caused severe defects in covalent adduct formation that correlated with the extent of inhibition of relaxation. None of these point mutations had an effect on noncovalent DNA binding sufficient to account for the defect in relaxation. Deletion of amino- or carboxyl-terminal portions of the polypeptide abrogated noncovalent DNA binding. Two distinct topoisomerase-DNA complexes were resolved by native gel electrophoresis. One complex, which was unique to those proteins competent in covalent adduct formation, contained topoisomerase bound to the 5'-portion of the incised DNA strand. The 3'-segment of the cleaved strand had dissociated spontaneously. This complex was isolated and shown to catalyze transfer of the covalently bound DNA to a heterologous acceptor oligonucleotide, thereby proving that the covalent adduct between protein and duplex DNA is a true intermediate in strand breakage and reunion. The role of the active site region of eukaryotic topoisomerase in determining sensitivity or resistance to camptothecin was examined by converting the active site region of the resistant vaccinia enzyme (SKRAY274) to that of the drug-sensitive yeast enzyme (SKINY). The SKINY mutation did not alter the resistance of the vaccinia enzyme to the cleavage-enhancing effects of camptothecin.

Amino Acid Sequence↗
PubMed1992

DNA strand transfer reactions catalyzed by vaccinia topoisomerase I.

Vaccinia virus DNA topoisomerase I forms a 3'-phosphoryl intermediate with duplex DNAs containing the conserved binding/cleavage motif 5'CCCTT decreases. Covalently bound enzyme is capable of transferring the incised DNA strand to a heterologous DNA acceptor containing a 5'OH terminus. Both intramolecular and intermolecular religation reactions are catalyzed. Intramolecular strand transfer occurs to the noncleaved strand of the DNA duplex and results in formation of a hairpin loop. Intermolecular religation to an exogenous DNA strand is favored over hairpin formation and requires the potential for base pairing between the acceptor and the noncleaved strand of the donor complex. As few as 4 potential base pairs are sufficient to support intermolecular transfer. These results in vitro are consistent with the proposal that vaccinia topoisomerase can catalyze sequence-specific strand transfer during genetic recombination in vivo (Shuman, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10104-10108.).

Autoradiography↗
PubMed1992

Stability of ternary transcription complexes of vaccinia virus RNA polymerase at promoter-proximal positions.

We have used DNA templates containing a vaccinia early promoter fused to G-less cassettes of varying length to study the formation of ternary transcription complexes by vaccinia virus RNA polymerase. Elongating polymerases were induced to pause at discrete sites on the DNA template by omission of GTP from transcription reactions. For most of the templates examined, the predominant sites of pausing were at or near the downstream border of the G-less transcription unit, as revealed by the size distribution of labeled RNAs synthesized in pulse-labeling reactions. Stability of ternary complexes containing nascent RNAs of any given length was assessed by the ability of these RNAs to be elongated upon provision of GTP. This criterion of stability could be met by complexes containing nascent RNAs as short as seven, eight, or nine nucleotides. In the presence of 3'-OMeGTP, nearly homogeneous populations of 3'-coterminal elongation complexes were positioned at the first G residue of the template. 3'-OMeG-arrested polymerases resumed elongation upon addition of GTP, apparently via sequential pyrophosphorolysis and nucleotide exchange at the site of elongation block. The ability to fix the 3' end facilitated analysis of initiation site choice based on the sizes of short nascent transcripts. Site choice was flexible and depended on the concentration of both the potential initiating NTP and the donor NTP participating in first phosphodiester bond formation. RNA polymerase could initiate at multiple positions within a nine-nucleotide region of the template. The rate of chain elongation by vaccinia polymerase during a single synchronous round of RNA synthesis was found to be 20-50 nucleotides per second.

Base Sequence↗
PubMed1992

A freeze-frame view of eukaryotic transcription during elongation and capping of nascent mRNA.

Ribonuclease footprinting of nascent messenger RNA within ternary complexes of vaccinia RNA polymerase revealed an RNA binding site that encompasses an 18-nucleotide RNA segment. The dimensions of the binding site did not change as the polymerase moved along the template. Capping of the 5' end of the RNA was cotranscriptional and was confined to nascent chains 31 nucleotides or greater in length. Purified capping enzyme formed a binary complex with RNA polymerase in solution in the absence of nucleic acid. These findings suggest a mechanism for cotranscriptional establishment of messenger RNA identity in eukaryotes.

Base Sequence↗
PubMed1992

Temperature-sensitive mutations in the vaccinia virus H4 gene encoding a component of the virion RNA polymerase.

Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts1, ts31, ts55, and ts58) comprising a single complementation group (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) have been mapped by marker rescue to the H4L open reading frame located within the genomic HindIII-H DNA fragment. The H4 gene is predicted to encode a 93.6-kDa polypeptide expressed at late times during infection. Nucleotide sequence alterations responsible for thermolabile growth lead to amino acid substitutions in the H4 gene product. All four ts alleles display "normal" patterns of early and late viral protein synthesis at the nonpermissive temperature (40 degrees C). Mature virion particles, microscopically indistinguishable from wild-type virions, are produced in the cytoplasm of cells infected with ts1 at 40 degrees C. Western immunoblot analysis localizes the H4 protein to the virion core. After solubilization from cores, the H4 protein is associated during purification with transcriptionally active vaccinia virus DNA-dependent RNA polymerase.

Amino Acid Sequence↗
PubMed1992

Ternary complex formation by vaccinia virus RNA polymerase at an early viral promoter: analysis by native gel electrophoresis.

We have resolved, by native gel electrophoresis, two intermediates in the transcription of a vaccinia virus early gene by the virus-encoded RNA polymerase. Polymerase holoenzyme containing the vaccinia virus early transcription factor (VETF) forms a complex of VETF bound to the promoter as the first step in a pathway leading to establishment of a committed ternary elongation complex. Formation of the VETF-DNA complex is stimulated by magnesium but is uninfluenced by nucleoside triphosphates. A stable binary complex of RNA polymerase bound to DNA is not detected. Assembly of a gel-stable polymerase-DNA complex depends on conditions permissive for RNA synthesis. Nucleotide omission experiments suggest that at least a tetrameric RNA must be made before a ternary complex is stabilized. RNA analysis indicates that complexes containing nascent transcripts 20 nucleotides long are stable and active. Ternary complex formation requires hydrolyzable ATP. This is consistent with an essential role for the ATPase activity of VETF at a step subsequent to DNA binding, as proposed by Broyles (S. S. Broyles, J. Biol. Chem. 266:15545-15548, 1991). The ternary complex, once formed, is resistant to dissociation by competitor DNA, as well as by salt, Sarkosyl, and heparin. The effects of these inhibitory agents on transcription complex formation suggest that they target different steps in the assembly pathway.

Base Sequence↗
PubMed1991

Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific.

Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi. As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se. The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA. Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage. To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced. In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners. Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage. Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision. The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.

Bacteriophage lambda↗
PubMed1991

Discrete functional stages of vaccinia virus early transcription during a single round of RNA synthesis in vitro.

We have developed a system for analysis of discrete steps in vaccinia virus early mRNA synthesis during a single round of transcription in vitro. A synthetic early promoter is used to direct transcription by vaccinia RNA polymerase of a G-less cassette in linear duplex DNA. Omission of GTP from transcription reactions leads to the formation of ternary elongation complexes paused stably at the end of the G-less cassette. These complexes can be induced to elongate by provision of GTP. While initiation of transcription is sensitive to low concentrations of salt and Sarkosyl, elongation is relatively resistant to these agents. Termination can be studied in a single synthetic cycle by forming transcription complexes paused just proximal to the termination signal TTTTTNT that can subsequently elongate and terminate. By selectively incorporating the termination-inhibiting analog BrUMP into proximal and distal portions of the nascent transcript, we localize the termination signal within or near the sequence UUUUUNU in the nascent RNA. We show that access of the vaccinia termination factor (VTF/capping enzyme) to the transcriptional apparatus can occur subsequent to initiation and synthesis of a 390-nucleotide nascent RNA. Termination is more sensitive to inhibition by salt and Sarkosyl than in elongation. This sensitivity is not reversed by preincubation of VTF with the transcription complex. Finally, we confirm the identity of VTF and vaccinia mRNA capping enzyme by demonstration of VTF activity associated with capping enzyme expressed in Escherichia coli.

Base Sequence↗