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 catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.
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PMID:Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding. 927 86

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.
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PMID:Intramolecular synapsis of duplex DNA by vaccinia topoisomerase. 935 38

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.
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PMID:Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease. 942 5

Molluscum contagiosum virus (MCV), the only member of the Molluscipoxvirus genus, causes benign papules in healthy people but disfiguring lesions in immunocompromised patients. The sequence of MCV has been completed, revealing that MCV encodes a probable type I topoisomerase enzyme. All poxviruses sequenced to date also encode type I topoisomerases, and in the case of vaccinia virus the topoisomerase has been shown to be essential for replication. Thus, inhibitors of the MCV topoisomerase might be useful as antiviral agents. We have cloned the gene for MCV topoisomerase, overexpressed and purified the protein, and begun to characterize its activities in vitro. Like other eukaryotic type I topoisomerases, MCV topoisomerase can relax both positive and negative supercoils. An analysis of the cleavage of plasmid and oligonucleotide substrates indicates that cleavage by MCV topoisomerase is favored just 3' of the sequence 5' (T/C)CCTT 3', resulting in formation of a covalent bond to the 3' T residue, as with other poxvirus topoisomerases. We identified solution conditions favorable for activity and measured the rate of formation and decay of the covalent intermediate. MCV topoisomerase is sensitive to inhibition by coumermycin A1 (50% inhibitory concentration, 32 microM) but insensitive to five other previously reported topoisomerase inhibitors. This work provides the point of departure for studies of the mechanism of function of MCV topoisomerase and the development of medically useful inhibitors.
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PMID:Molluscum contagiosum virus topoisomerase: purification, activities, and response to inhibitors. 952 70

Vaccinia DNA topoisomerase breaks and rejoins DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A C-terminal catalytic domain, Topo(81-314), suffices for transesterification chemistry. The domain contains a constellation of five amino acids, conserved in all eukaryotic type IB topoisomerases, that catalyzes attack of the tyrosine nucleophile on the scissile phosphate. The structure of the catalytic domain, consisting of ten alpha helices and a three-strand beta sheet, resembles the catalytic domains of site-specific recombinases that act via a topoisomerase IB-like mechanism. The topoisomerase catalytic pentad is conserved in the tertiary structures of the recombinases despite scant sequence similarity overall. This implies that the catalytic domains of type IB topoisomerases and recombinases derive from a common ancestral strand transferase.
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PMID:Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases. 952 59

The metabolic fate of covalently linked DNA-protein complexes (cross-links) is not clearly understood. Our aim was to investigate the processing of protein-DNA cross-links by cellular enzymes. As an example of a DNA-protein cross-link, we have constructed frozen topoisomerase-DNA conjugates and investigated their processing by human cell-free extracts. A suicide DNA substrate was constructed that upon reaction with vaccinia type I topoisomerase yielded a highly stable covalent DNA-protein cross-link. When this conjugate was treated with human nuclear or whole cell extracts, two sites of DNA breakpoints were detected: one set of double-stranded breaks occurred close to the 3' side of the topoisomerase (topo) conjugation site, and there was another set of nicks about 30 nucleotides 3' to the topo site. The double-stranded breaks were not made by extracts from xeroderma pigmentosum group A mutant cells, suggesting that the xeroderma pigmentosum group A damage recognition protein may be required for the occurrence of DNA breakage. In addition to these DNA breakage reactions, there was an activity that resulted in the delinking of the frozen topoisomerase (or proteolytic fragments thereof) from the DNA substrate, which was followed by a ligation step that restored the continuity of the broken DNA strand at the erstwhile topo attachment site. We suggest that frozen topoisomerase-DNA conjugates (and perhaps other types of covalent DNA-protein complexes) are processed by multiple pathways that may involve the cleavage of the DNA in the covalent protein-DNA complex and/or enzymatic delinking followed by ligation of the broken DNA ends. These processes may represent the "repair" of DNA-protein cross-links.
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PMID:Mechanisms for the processing of a frozen topoisomerase-DNA conjugate by human cell-free extracts. 954 38

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.
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PMID:A catalytic domain of eukaryotic DNA topoisomerase I. 956 76

The recently determined crystal structures of fragments of the human and vaccinia virus type IB topoisomerases reveal unexpected similarity with the lambda family of site-specific recombinases. The conservation of structure suggests a common mechanism, indicating that topoisomerase activity may be the consequence of uncoupling DNA strand cleavage/religation from synapsis.
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PMID:Teaching a new dog old tricks? 963 92

Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein-DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3'-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2' OH of the ribose sugar to yield a free 2', 3' cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.
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PMID:Site-specific ribonuclease activity of eukaryotic DNA topoisomerase I. 965 6

Introduction of a single ribonucleoside immediately 5' of the scissile phosphate of a duplex DNA substrate converts eukaryotic topoisomerase I into an endoribonuclease. Here, I demonstrate that the RNase reaction is reversible. Vaccinia topoisomerase can ligate 2', 3'-cyclic phosphate and 5'-hydroxyl termini annealed to a bridging template strand. Remarkably, the ligase activity of topoisomerase does not require the active site tyrosine, implying that strand joining can occur via direct attack of the 5' hydroxyl on the cyclic phosphate without a covalent intermediate. Ligation does require other catalytic side chains on the enzyme. These findings underscore how a common ancestral mechanism of phosphoryl and nucleotidyl transfer can be harnessed to perform seemingly diverse tasks through subtle changes at the active site.
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PMID:Polynucleotide ligase activity of eukaryotic topoisomerase I. 966 Sep 57


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