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

Binding to DNA and synthetic duplex polymers of two bifunctional netropsins and effects on supercoiled plasmid DNA as well as their inhibitory potency on DNA topoisomerases have been investigated. Characteristic differences were found in the DNA binding properties of the two bis-netropsins containing a cis and trans tether as reflected by CD, thermal melting and sedimentation measurements. CD results indicate, that the bis-netropsins interact with DNA by a two-step binding mode depending on the ligand concentration. The trans bis-netropsin may form stable complexes with different DNA's at high salt concentration, whereas for cis bis-netropsin DNA complexes the second binding step is completely abolished. The variations in the DNA binding ability of trans and cis bis-netropsin show a close relationship to the differences observed in their inhibitory effects on DNA topoisomerases. It appeared that trans bis-netropsin more strongly blocks topoisomerase activity than the cis isomer and represents the most potent inhibitor of DNA gyrase. Differences in the DNA. binding ability of the bis-netropsins and their inhibitory potency on topoisomerase activity are explained in terms of bidentate and monodentate binding mode of the trans and cis isomer, respectively.
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PMID:DNA binding studies and influence on the activity of DNA topoisomerases of bis-netropsins: different effects of analogs containing cis and trans ethylene linkers. 928 82

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.
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PMID:Identification of DNA replication and cell cycle proteins that interact with PCNA. 939 13

S16020-2 (NSC-659687) is a new olivacine derivative that is highly cytotoxic in vitro and displays remarkable antitumor activity against various experimental tumors, especially some solid tumor models. Its antitumor activity is notably higher than that of 2-methyl-9-hydroxy-ellipticinium (NMHE) and comparable to that of doxorubicin HCl, although with a different tumor specificity. S16020-2 is being tested in phase I clinical trials. A study of the interaction of S16020-2 with DNA showed that it binds through intercalation between adjacent DNA base pairs, inducing an unwinding of 10 degrees of the double helix. Its DNA affinity is approximately equal to that of NMHE and decreases as a function of the salt concentration, indicating a significant electrostatic contribution to the overall binding free energy. S16020-2 did not interfere with the catalytic cycle of DNA topoisomerase I but stimulated DNA topoisomerase II-mediated DNA cleavage via a strictly ATP-dependent mechanism. The interactions of S16020-2 and NMHE with DNA topoisomerase II in vitro are very similar. Both drugs have the same DNA sequence specificity of cleavage and the same biphasic dose-effect response, and neither drug inhibited the rate of DNA religation. In contrast with these observations, in in vivo experiments, S16020-2 was able to induce topoisomerase II-mediated DNA strand breaks at concentrations 500-fold lower than NMHE. We conclude that DNA topoisomerase II most likely is the cellular target involved in the mechanism of cytotoxicity of S16020-2. Its higher biological activity and potency to induce cellular DNA cleavage suggest the involvement of as-yet-unidentified cellular factors.
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PMID:S16020-2, a new highly cytotoxic antitumor olivacine derivative: DNA interaction and DNA topoisomerase II inhibition. 946 78

We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase IIalpha isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase IIalpha in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase IIalpha isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.
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PMID:In vivo etoposide-resistant C6 glioma cell line: significance of altered DNA topoisomerase II activity in multi-drug resistance. 952 24

Substituting Lys359 with either Gln or Glu in the highly conserved QTK-loop in the DNA gyrase B protein homologous domain of Drosophila topoisomerase II inactivates its catalytic activities. Although strand passage and DNA-dependent ATPase activities are affected in these mutant proteins, their DNA cleavage activity is comparable with the wild-type enzyme and can be stimulated to the same level by topoisomerase-targeting anticancer drugs. The sequence specificity in the DNA cleavage reaction remains unaltered for the mutant proteins. We have used both glass fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both Gln and Glu mutant proteins can form a clamp complex in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, albeit with a lower efficiency than the wild-type enzyme. However, the mutant proteins can form a stable complex either in the presence of ATP or in the absence of any cofactors. These results are in an interesting contrast with the wild-type enzyme, which cannot form a stable complex under similar conditions. Our data suggest that Lys359 is critical for the catalytic activity of topoisomerase II and may have an important function in the ATP signaling process.
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PMID:Identifying Lys359 as a critical residue for the ATP-dependent reactions of Drosophila DNA topoisomerase II. 954 89

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

Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.
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PMID:Step-wise DNA relaxation and decatenation by NaeI-43K. 958 Jun 89

The possibility to record a trace of the precise sites of topoisomerase action has been exploited for almost 12 years in many laboratories. The large majority of the studies were performed in vitro, giving a good picture of sequence specificities of topoisomerases, and of the preference of various drugs for some sequences. Only a relatively small number of reports concern in vivo studies. Their main conclusions are the following: i) topoisomerase II sites are often found near replication origins and termini, where they are supposed to play a role in the decatenation of daughter DNA molecules, and possibly in the initiation of replication; ii) topoisomerase II sites are found in the promoter region of many genes, but they seem related to the condensation state of chromatin in this region, rather than to transcription per se; iii) some topoisomerase II sites, resistant to high salt, are found in or near matrix associated regions (MARs), suggesting a role in loop anchorage or (and) in the control of topology of individual chromatin loops; iv) topoisomerase I sites appear less localized, acting all along the transcription units, where they seem directly involved in transcription; and v) topoisomerase I sites are possibly connected with replication fork progression and (or) with the termination of replication. Despite these advances, the precise role of topoisomerases in vivo is still poorly understood, especially in recombination and chromatin condensation and decondensation during the cell cycle. Future attempts should take into account the possible specialization of the multiple topoisomerases found in a given cell, and the use of highly synchronized systems.
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PMID:The mapping of DNA topoisomerase sites in vivo: a tool to enlight the functions of topoisomerases. 961 62

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.
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PMID:Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159. 967 16


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