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)

The eukaryotic family of type I DNA topoisomerases includes the nuclear type I enzymes and the enzymes encoded by vaccinia and other poxviruses. The small size of the vaccinia topoisomerase (314 amino acids as compared to 765-972 amino acids for the cellular enzymes) makes it likely that this protein constitutes the minimal functional unit of a eukaryotic type I enzyme and provides an opportunity for a comprehensive structure-function analysis through mutagenesis. Two protein subregions were targeted for mutagenesis in the present study. The role of the Ser-Lys-X-X-Tyr sequence present at the active site of all family members was examined by replacing each conserved residue with alanine. Alanine substitution at the active site Tyr abrogated topoisomerase activity. In contrast, mutations at Ser-270 and Lys-271 had no effect on enzyme activity. The region of the vaccinia topoisomerase from amino acids 126-142 (MFFIRFGKMKYLKENET) is highly conserved and contains a residue, Gly-132, shown previously to be essential. Twenty-nine different mutations were generated in this region, with at least one substitution at each position. Point mutations were identified at three positions, Arg-130, Tyr-136, and Leu-137, which either abrogated or severely reduced DNA relaxation. The effects on activity could be attributed to a defect in formation of the covalent intermediate. Alterations of 13 other amino acids, including conserved residues, had little or no effect on topoisomerase activity.
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PMID:Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis. 796 97

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

Site-specific recombination catalyzed by bacteriophage lambda integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the proteins are involved in protein-protein contacts that control the timing of DNA cleavage events during recombination. We investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Alanine scanning mutagenesis of the C-terminus indicates that two mutants, W350A and I353A, cannot perform site-specific recombination although their DNA binding, cleavage and ligation activities are at wild-type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA. To explain these results, we have constructed a homology-threaded model of the Int structure using a Cre crystal structure. We propose that residues R346 and R348 are involved in orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the C-terminus.
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PMID:Regulation of site-specific recombination by the C-terminus of lambda integrase. 1246 44

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp(168) is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.
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PMID:Regulation of catalysis by the smallpox virus topoisomerase. 1703 43