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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin. 856 95

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.
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PMID:Resolution of Holliday junctions by eukaryotic DNA topoisomerase I. 857 Jun 35

Several fowlpox virus (FPV) DNA fragments were selected by differential hybridization using cDNA of transcripts that were strongly transcribed early and/or later after infection of QT-35 cells. The EcoRI L fragment contained three strongly transcribed FPV genes: L1L, a late 1452 bp partial (amino end) ORF; L2R, an early/late 522 bp ORF; and L3R, a late 948 bp ORF. The protein products of L1L, L2R and L3R shared homology with the products of vaccinia virus (VV) genes H4L (RAP94), H5R (Ag35) and H6R (topoisomerase), respectively, suggesting a conservation of gene structure and order between VV and FPV. The 5' upstream non-coding sequences of L1L and L3R were A + T rich and the sequence 5' TAAATG 3' overlapped the predicted translation start codon. Primer extension analysis of the L2R transcript mapped the transcriptional start sites of early and late mRNAs 14 nt downstream of a VV early promoter-like critical region sequence, AAAATTGAA-AAAAAAA. A VV-like TAAAT late transcriptional element was present 20 nt upstream of the L2R ATG translational start codon. A plasmid with the putative early L2R promoter cloned upstream of the Newcastle disease virus haemagglutinin-neuraminidase (HN) cDNA as a reporter gene was at least 6-fold more effective in generating HN MRNa than plasmids containing the P7.5 or P11 VV promoters in transient expression assays in FPV-infected CEF cells treated with cytosine arabinoside. The L2R promoter was also able to express an amount of HN mRNA equal to that expressed by the VV promoters late in infection.
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PMID:Partial transcriptional mapping of the fowlpox virus genome and analysis of the EcoRI L fragment. 862 48

Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH2-terminal domain (approximately 24 kDa), which is known to be dispensable for activity, (ii) the core domain (approximately 54 kDa), (iii) a linker region (approximately 3 kDa), and (iv) the COOH-terminal domain (approximately 10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH2-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH2-terminal domain. When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive charge of the linker domain suggests that at a certain point in catalysis the linker may bind directly to DNA. Further, we show that limited subtilisin cleavage can generate a mixture of 60-kDa core and approximately 10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH2-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies.
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PMID:The domain organization of human topoisomerase I. 863 94

Poxviral DNA topoisomerases are sequence-specific enzymes whose activities are thought to influence such diverse processes as transcription, DNA replication, and genetic recombination. To obtain further insights into the relatedness of these enzymes, and their influence on virus-mediated recombination, we have determined the target-specificity and other catalytic properties of the Shope fibroma virus (SFV) topoisomerase. SFV topoisomerase was expressed in Escherichia coli and purified as a glutathione S-transferase (GST) or (his)6-tagged fusion protein. The recombinant Leporipox-virus (SFV) enzyme displayed catalytic properties very similar to vaccinia topoisomerase. In particular SFV topoisomerase recognizes the same pentanucleotide motif [5'-(C/T)CCTT-3'] and promotes the same DNA relaxation, strand transfer, and strand cleavage reactions catalyzed by the Orthopoxviral (vaccinia) enzyme. The SFV enzyme can also efficiently cleave DNA 3' of the variant site 5'-CCCTG-3' in certain sequence contexts. These studies identified several sites where SFV topoisomerases interact with a recombinational substrate and permitted a comparison of recombination frequencies across intervals which did, or did not, span these sites. We failed to detect any effect of topoisomerase-recognition sites on recombination frequencies, except for a small (< 2-fold) stimulation seen when the substrates encoded a nearby poxviral promoter. This and other work shows that poxviral topoisomerases from several genera share common target specificities, but other enzymatic systems probably catalyze the high-frequency recombination seen in poxvirus-infected cells.
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PMID:SFV topoisomerase: sequence specificity in a genetically mapped interval. 866 46

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, binds and cleaves duplex DNA at sites containing the sequence 5'-(T/C)CCTT. We report the identification of Tyr70 as the site of contact between the enzyme and the +4C base of its target site. This was accomplished by UV-crosslinking topoisomerase to bromocytosine-substituted DNA, followed by isolation and sequencing of peptide-DNA photoadducts. A model for the topoisomerase-DNA interface is proposed, based on the crystal structure of a 9 kDa N-terminal tryptic fragment. The protein domain fits into the DNA major groove such that Tyr70 is positioned close to the +4C base and Tyr72 is situated near the +3C base. Mutational analysis indicates that Tyr70 and Tyr72 contribute to site recognition during covalent catalysis. We propose, based on this and other studies of the vaccinia protein, that DNA backbone recognition and reaction chemistry are performed by a relatively well-conserved 20 kDa C-terminal portion of the vaccinia enzyme, whereas discrimination of the DNA sequence at the cleavage site is accomplished by a separate N-terminal domain, which is less conserved between viral and cellular proteins. Division of function among distinct structural modules may explain the different site specificities of the eukaryotic type I topoisomerases.
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PMID:Identification of contacts between topoisomerase I and its target DNA by site-specific photocrosslinking. 867 Aug 47

We have used potassium permanganate to probe contacts between vaccinia DNA topoisomerase and thymine residues in its 5'-CCCTT downward arrow DNA target site. Two major conclusions emerge from the experiments presented: (i) permanganate oxidation of the +2T base of the scissile strand interferes with topoisomerase binding to DNA, and (ii) the +1T base of the scissile strand becomes unpaired upon formation of the covalent topoisomerase-DNA intermediate. Disruption of T:A base pairing is confined to the +1-position. Covalently bound DNAs that have experienced this structural distortion (such DNAs being marked by oxidation at +1T) are fully capable of being religated. We suggest that a protein-induced DNA conformational change is a component of the strand passage step of the topoisomerase reaction.
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PMID:Covalent DNA binding by vaccinia topoisomerase results in unpairing of the thymine base 5' of the scissile bond. 870 32

The purpose of this review is to summarize information published since 1990 on DNA replication, recombination and repair of vaccinia virus, a poxvirus. Temperature-sensitive mutations reveal four essential genes related to viral DNA replication: the E9L DNA polymerase, B1R protein kinase, D5R protein, and D4R uracil DNA glycosylase. Other proteins are likely to be also involved in viral DNA replication: the H6R DNA topoisomerase, I3L single stranded-DNA binding protein, H5R virosome-associated protein, and A50R DNA ligase. In addition, several viral-encoded proteins do regulate the level of the deoxyribonucleoside triphosphate pool: the J2R thymidine kinase, A48R thymidylate kinase, 14L and F4L subunits of ribonucleotide reductase, and F2L dUTPase. Despite the apparent simplicity of the mechanism of vaccinia virus DNA replication, several important questions related to the three Rs remain unsolved.
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PMID:Vaccinia virus DNA replication: a short review. 882 74

The segment of the vaccinia DNA topoisomerase from residues 143 to 167 (VGLLTLKNKHIEISPDEIVIKFVGK) is conserved in other members of the eukaryotic type I topoisomerase family. In order to gauge the function of this region, we performed a mutational analysis in which 23 of 25 positions were substituted by alanine. Several non-alanine mutations were also studied. Purified wild-type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA and in single-turnover strand cleavage. Lys167, an invariant residue, was judged essential for catalysis, insofar as alanine replacement resulted in a 100-fold decrement in specific activity. Alanine substitutions for invariant residues Gly144 and Gly166 were well-tolerated, but a G144R mutation inactivated the enzyme and G166R reduced activity by two orders of magnitude. More modest effects of other mutations were demonstrated by kinetic analysis of the single-turnover DNA cleavage and religation reactions and by studies of covalent adduct formation under equilibrium conditions. Mutations G144A and T147A elicited a shift in the cleavage-religation equilibrium toward the non-covalently bound state; this was caused by slowing of the forward cleavage reaction. Mutations F164A, G166A, G166R, K167A, and K167R produced opposite effects on reaction equilibrium, resulting in higher levels of covalent complex formation. We suggest that invariant residues F164, G166, and K167, constitute part of the active site of the enzyme.
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PMID:Mutations within a conserved region of vaccinia topoisomerase affect the DNA cleavage-religation equilibrium. 891

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.
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PMID:Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription. 897 Sep 79


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