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)

DNA topoisomerases II are enzymes which have been purified from a lot of organisms and have been found to be involved in segregation of chromosomes. The following article reports the analysis of a partially purified DNA topoisomerase II from Sulfolobus (strain B12) a thermophilic archaebacterium which grows at 80 degrees C. The enzyme is composed by two subunits: the A subunit with a molecular mass of 85,000 Da which contains the nicking-closing activity and the B subunit with a molecular mass of 65,000 Da which contains the ATP binding site. The enzyme relaxes negatively as well as positively supercoiled DNA consequently to ATP hydrolysis into ADP (as eukaryotic DNA topoisomerases II and Escherichia coli DNA topoisomerase IV do). DNA relaxation catalyzed by the thermophilic enzyme is inhibited in the presence of both bacterial antibiotics acting at the ATP binding site such as novobiocin and coumermycin A1 at the concentration which was found to inhibit the E. coli type II DNA topoisomerases (DNA gyrase and DNA topoisomerase IV). Based on the relaxation of both negatively and positively supercoiled DNA and the sensitivity to antibiotics such as novobiocin and coumermycin A1, the DNA topoisomerase II isolated from thermophilic archaebacterium shares common characteristics with E. coli DNA topoisomerase II.
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PMID:DNA unknotting activity (DNA topoisomerase II) isolated from a thermophilic archaebacterium Sulfolobus is inhibited by novobiocin. Partial purification, identification of the two subunits and characteristics of the enzyme. 808 47

Sulfolobus shibatae B12 is a thermophilic archaebacterium that contains an inducible virus named SSV1. The viral DNA has been shown to be positively supercoiled before encapsidation. We have previously purified an archaebacterial DNA topoisomerase from Sulfolobus acidocaldarius DSM 639, reverse gyrase, likely responsible for this positive supercoiling reaction. In order to study an homogeneous system containing both reverse gyrase and one of its preferential substrate, SSV1 DNA, we have purified this enzyme from S. shibatae. During the course of the purification, we have detected another topoisomerase activity. In order to separate and purify these two topoisomerases, we have devised a new purification procedure. Purified S. shibatae reverse gyrase is a 124-kDa monomer, with a Stokes radius of 43 A and a sedimentation coefficient of 6.2 S. It is able to perform a DNA reverse gyration per se at 10 mM NaCl in a Mg- and ATP-dependent manner. The other topoisomerase is a monomer of about 40 kDa, with a Stokes radius of 25 A and a sedimentation coefficient of 4 S. This additional topoisomerase activity is Mg-dependent and ATP-independent and catalyzes only a relaxation reaction of negatively supercoiled DNA at 150 mM NaCl. This new ATP-independent topoisomerase activity seems to be a proteolysis product of reverse gyrase.
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PMID:Purification and characterization of reverse gyrase from Sulfolobus shibatae. Its proteolytic product appears as an ATP-independent topoisomerase. 810 9

Merbarone is a catalytic inhibitor of DNA topoisomerase (topo) II that does not stabilize DNA-topo II cleavable complexes. Although the cytotoxicity of and resistance to complex-stabilizing topo II inhibitors, such as etoposide, is thought to be mediated through stabilization of these complexes, the mechanisms of cytotoxicity and resistance to catalytic inhibitors are not well known. To investigate this issue, we established 12 merbarone-resistant cell lines from human leukemia CEM cells, designated CEM/M70-B1 through -B12. Assessed by either growth inhibition or clonogenic assay, these cell lines are 3.5- to 6.6-fold resistant to merbarone, compared to the CEM parent cells. Karyotype analysis of three of the cell lines revealed that while CEM and drug-resistant cell lines had chromosome abnormalities in common, indicating a common origin, two of the merbarone-resistant lines (B1 and B8) each had unique structural markers. These novel cell lines are cross-resistant to complex-stabilizing topo II inhibitors, etoposide, teniposide, amsacrine, and doxorubicin, but not to other catalytic inhibitors, aclarubicin or SN-22995. Of considerable interest, these cell lines are cross-resistant to SN-38, a putative topo I inhibitor, but cross-resistance to other topo I inhibitors (camptothecin and topotecan) was lower and not seen in every cell line. In all 12 cell lines, there was a high correlation among drug resistance ratios between etoposide and teniposide and between merbarone and SN-38. By contrast, there was a low correlation between merbarone and etoposide and between SN-38 and other topo I inhibitors. These results suggest that resistance to merbarone and cross-resistance to etoposide might be through different mechanisms, whereas cross-resistance to SN-38 might be through a merbarone-related mechanism. Etoposide and SN-38 stabilized fewer DNA-topoisomerase complexes in CEM/M70-B cells than in CEM cells, but camptothecin stabilized more. Merbarone inhibited complex formation induced by etoposide in drug-sensitive and -resistant cells, but the degree of inhibition was lower in CEM/M70-B cells than in the parental cells. Moreover, merbarone did not affect complex formation stabilized by SN-38 or camptothecin. Immunoblot analysis of the CEM/M70-B cells showed decreased topo IIalpha, increased topo IIbeta, and no change of topo I protein, compared to CEM cells. We propose the hypothesis that decreased topo IIalpha may play a role in the resistance to merbarone that is different from that to complex-stabilizing drugs. Cross-resistance to catalytic inhibitors may be due to reduced complex formation as a consequence of decreased topo IIalpha. We also found that DNA-protein complexes stabilized by SN-38 might be different from those stabilized by topo II inhibitors and blocked by merbarone. Judging from both the high correlation of drug sensitivities and complex-formation assays, we postulate that mechanisms of cytotoxicity and cross-resistance of SN-38 in CEM/M70-B cells might be similar to those of merbarone. We believe that the CEM/M70-B cells are the first to be selected and characterized for resistance to a catalytic inhibitor of topo II. This study provides novel cell lines with characteristics of resistances to topo II and topo I inhibitors.
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PMID:Characterization of novel human leukemic cell lines selected for resistance to merbarone, a catalytic inhibitor of DNA topoisomerase II. 865

We cloned and sequenced a DNA fragment from the thermophilic archaeal strain Sulfolobus shibatae B12 that includes the gene topR encoding the reverse gyrase. The RNA of the reverse gyrase gene was characterized indicating that the topR gene is fully functional in vivo. We showed by primer extension analysis that transcription of topR initiates 28 bp downstream from a consensus A-box promoter. In order to understand how this particular type I DNA topoisomerase introduces positive superturns into the DNA, we compared the amino acid sequence of reverse gyrase from S.shibatae with the two other known reverse gyrases. This comparison indicates a common organization of these proteins: the carboxy-terminal domain is related to the type I-5' topoisomerase family while the amino-terminal domain possesses some motifs of proteins described as RNA or DNA helicases. By using local alignments, we showed that (i) reverse gyrases constitute a new and rather homogenous group within the type I-5' DNA topoisomerase family; (ii) a careful sequence analysis of the amino-terminal domain allows us to relate the presence of some motifs with an ATP binding and hydrolysis reaction coupled to a DNA binding and unwinding activity.
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PMID:Reverse gyrase gene from Sulfolobus shibatae B12: gene structure, transcription unit and comparative sequence analysis of the two domains. 897 52

Reverse gyrase is a type I-5' topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes places, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA-S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate increases it without changing the sequence specificity.
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PMID:Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12. 1009 89