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 topoisomerase catalyzes DNA cleavage and rejoining via transesterification to pentapyrimidine recognition site 5'-(C/T)CCTT downward arrow in duplex DNA. The proposed reaction mechanism involves general-base catalysis of the attack by active site nucleophile Tyr-274 on the scissile phosphodiester and general-acid catalysis of the expulsion of the 5'-deoxyribose oxygen on the leaving DNA strand. The pKa values suggest histidine and cysteine side chains as candidates for the roles of proton acceptor and donor, respectively. To test this, we replaced each of the eight histidines and two cysteines of the vaccinia topoisomerase with alanine. Single mutants C100A and C211A and a double mutant C100A-C211A were fully active in DNA relaxation, indicating that a cysteine is not the general acid. Only one histidine mutation, H265A, affected enzyme activity. The rates of DNA relaxation, single-turnover strand cleavage, and single-turnover religation by H265A were 2 orders of magnitude lower than the wild-type rates. Yet the H265A mutation did not alter the dependence of the cleavage rate on pH, indicating that His-265 is not the general base. Replacing His-265 with glutamine or asparagine slowed DNA relaxation and single-turnover cleavage to about one-third of the wild-type rate. All three mutations, H265A, H265N, and H265Q, skewed the cleavage-religation equilibrium in favor of the covalently bound state. His-265 is strictly conserved in every member of the eukaryotic type I topoisomerase family.
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PMID:Histidine 265 is important for covalent catalysis by vaccinia topoisomerase and is conserved in all eukaryotic type I enzymes. 902 90

Vaccinia DNA topoisomerase, a 314-amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. To identify amino acids that participate in the transesterification reaction, we introduced alanine substitutions at 39 positions within a conserved 57amino acid segment upstream of the active-site tyrosine. Purified wild type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA. The majority of mutant proteins displayed wild type topoisomerase activity. Mutant enzymes that relaxed DNA at reduced rates were subjected to kinetic analysis of the strand cleavage and religation steps under single-turnover and equilibrium conditions. For the wild type topoisomerase, the observed single-turnover cleavage rate constant (kcl) was 0.29 s-1 and the cleavage-religation equilibrium constant (Kcl) was 0.22. The most dramatic mutational effects were seen with R223A; removal of the basic side chain reduced the rates of cleavage and religation by factors of 10(-4.3) and 10(-5.0), respectively, and shifted the cleavage-religation equilibrium in favor of the covalently bound state (Kcl = 1). Introduction of lysine at position 223 restored the rate of cleavage to 1/10 that of the wild type enzyme. We conclude that a basic residue is essential for covalent catalysis and suggest that Arg-223 is a constituent of the active site. Modest mutational effects were observed at two other positions (Lys-220 and Asn-228), at which alanine substitutions slowed the rates of strand cleavage by 1 order of magnitude and shifted the equilibrium toward the noncovalently bound state. Arg-223 and Lys-220 are conserved in all members of the eukaryotic type I topoisomerase family; Asn-228 is conserved among the poxvirus enzymes.
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PMID:Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis. 907 46

Vaccinia topoisomerase relaxes DNA through the formation of a covalent DNA-(3'-phosphotyrosyl)protein intermediate at sites containing the sequence 5'-(T/C)CCTT/. The active site, Tyr-274, is situated near the carboxyl terminus of the 314 amino acid enzyme. Here, we report the effects of serial C-terminal deletions. Removal of five amino acids had no effect on topoisomerase activity. However, deletion of 10, 15, or 20 amino acids rendered the enzyme distributive in DNA relaxation, incrementally slowed the rate of single-turnover DNA cleavage, and progressively diminished DNA binding affinity, without altering the sequence specificity of DNA cleavage. These effects lead us to speculate that the region downstream of the active site, which is not well-conserved among the poxvirus-encoded topoisomerases, is a component of the proposed circumferential interface between the enzyme and duplex DNA.
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PMID:Deletions at the carboxyl terminus of vaccinia DNA topoisomerase affect DNA binding and enhance distributivity in DNA relaxation. 909 21

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release: (I) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilibrium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (approximately 20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (delta n = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.
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PMID:Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation. 913 83

We have identified an Amsacta moorei entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT decreases. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase (Kobs = 0.08 sec-1; Kcl = 0.22) are similar to those of the vaccinia virus enzyme.
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PMID:Characterization of a DNA topoisomerase encoded by Amsacta moore entomopoxvirus. 914 75

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.
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PMID:DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate. 915 7

In order to study the mechanisms by which p53 function is regulated, human wild-type p53 cDNA was cloned into a vaccinia virus vector and the expressed p53 protein was used to investigate binding of the p53 by cellular proteins from a cDNA expression library from human liver. One protein that bound wild-type p53 had > 99% homology with DNA topoisomerase IIb. p53 protein was coimmunoprecipitated from topoisomerase II-rich cell lysates (but not from topoisomerase II-deficient cell lysates) by an antibody to topoisomerase IIa and IIb. This binding was shown to occur without a dsDNA intermediary. Hepatocellular carcinomas (HCCs) and adjacent nontumorous liver tissues from ten patients were studied to determine the level of expression of topoisomerase II and p53. Overexpressed topoisomerase II proteins were detected by western blot in six of ten HCCs (60%), including several in which presumed wild-type p53 was detected by immunohistochemistry. No topoisomerase II expression was detectable in the ten nontumorous liver tissues from the same patients or in a sample of normal human liver.
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PMID:Binding of wild-type p53 by topoisomerase II and overexpression of topoisomerase II in human hepatocellular carcinoma. 916 88

Vaccinia topoisomerase binds duplex DNA and forms a covalent DNA-(3'-phosphotyrosyl) protein adduct at the sequence 5'-CCCTT downward arrow. The enzyme reacts readily with a 36-mer CCCTT strand (DNA-p-RNA) composed of DNA 5' and RNA 3' of the scissile bond. However, a 36-mer composed of RNA 5' and DNA 3' of the scissile phosphate (RNA-p-DNA) is a poor substrate for covalent adduct formation. Vaccinia topoisomerase efficiently transfers covalently held CCCTT-containing DNA to 5'-OH-terminated RNA acceptors; the topoisomerase can therefore be used to tag the 5' end of RNA in vitro. Religation of the covalently bound CCCTT-containing DNA strand to a 5'-OH-terminated DNA acceptor is efficient and rapid (krel > 0.5 s-1), provided that the acceptor DNA is capable of base pairing to the noncleaved DNA strand of the topoisomerase-DNA donor complex. The rate of strand transfer to DNA is not detectably affected by base mismatches at the 5' nucleotide of the acceptor strand. Nucleotide deletions and insertions at the 5' end of the acceptor slow the rate of religation; the observed hierarchy of reaction rates is as follows: +1 insertion > -1 deletion > +2 insertion >> -2 deletion. These findings underscore the importance of a properly positioned 5'-OH terminus in transesterification reaction chemistry, but they also raise the possibility that topoisomerase may generate mutations by sealing DNA molecules with mispaired or unpaired ends.
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PMID:Kinetic analysis of DNA and RNA strand transfer reactions catalyzed by vaccinia topoisomerase. 918 65

Vaccinia DNA topoisomerase, a 314 amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. To identify amino acids that participate in the DNA binding and transesterification steps, we introduced alanine substitutions at 18 positions within a centrally located 27 amino acid segment (181-RLYKPLLKLTDDSSPEEFLFNKLSERK-207) and at 8 positions near the N-terminus (1-MRALFYKDGK-10). All mutant proteins except two displayed wild-type activity in relaxing supercoiled DNA. F200A and S204A exhibited reduced rates of relaxation and were subjected to a kinetic analysis of the strand cleavage reaction under single-turnover and equilibrium conditions. The F200A and S204A mutations reduced the rate of single-turnover DNA cleavage by factors of 5 and 70, respectively. Both mutations shifted the cleavage-religation equilibrium in favor of the noncovalently bound state. The S204A mutation reduced the affinity of topoisomerase for CCCTT-containing DNA, but did not alter the site-specificity of DNA cleavage. Vaccinia residue Ser-204, which is conserved in all poxvirus topoisomerases, but not in the cellular homologues, may contribute to the unique cleavage site specificity of the poxvirus enzymes. Phe-200 is conserved in all members of the type IB topoisomerase family.
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PMID:Mutational analysis of 26 residues of vaccinia DNA topoisomerase identifies Ser-204 as important for DNA binding and cleavage. 920 40

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.
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PMID:Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167. 922 99


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