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)

Plumbagin and shikonin, plant metabolites which have naphthoquinone structures, induced mammalian topoisomerase II-mediated DNA cleavage in vitro. Treatment of a reaction mixture containing these naphthoquinones and topoisomerase II at an elevated temperature (65 degrees C) resulted in a great reduction in DNA cleavage, suggesting that the mechanism of the topoisomerase II-mediated DNA cleavage induced by these naphthoquinones is through formation of a cleavable complex, as seen with antitumor agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide and demethylepipodophyllotoxin ethylidene-beta-glucoside. Lawson and lapacol, which are structurally related plant metabolites with naphthoquinone moieties, could not induce topoisomerase II-mediated DNA cleavage. Plumbagin and shikonin induced a similar DNA cleavage pattern with topoisomerase II which was different from the cleavage patterns induced with other known topoisomerase II-active drugs. A DNA-unwinding assay with T4 DNA ligase showed that shikonin, lawson, and lapacol did not intercalate into DNA, while plumbagin and 2-methyl-1,4-naphthoquinone intercalate into DNA, but to a lower degree than 4'-(9-acridinylamino)methanesulfon-m-anisidide does.
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PMID:Induction of topoisomerase II-mediated DNA cleavage by the plant naphthoquinones plumbagin and shikonin. 133 38

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.
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PMID:Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus. 164 1

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

The specificities of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase from brain and liver cells of the chick embryo and of DNase I were demonstrated in vitro by their response to substrate DNA pretreated with monofunctional alkylating agents of different O6-guanine alkylating ability and some antineoplastic agents. Treatment of DNA with ethidium bromide, Hoechst 33258, doxorubicin, Fe2+/bleomycin, and suramin resulted in a dose-dependent diminution of alkyltransferase activity (DE50 approximately 5 micrograms/ml, 15 micrograms/ml, 5 micrograms/ml, 5 micrograms/ml, 100 micrograms/ml, respectively). Apart from bleomycin, comparable results were obtained with DNase I. Thermal denaturation of the substrate DNA reduced both alkyltransferase and DNase I activity. No effect was seen with X-irradiation. Cisplatin decreased only DNase I activity. Some topoisomerase II and/or gyrase inhibitors remained without significant effects on the alkyltransferase reaction whereas DNA catabolism by DNase I was diminished in a dose-dependent manner (DE50 between 6.5 and 19 micrograms/ml).
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PMID:Inhibition of O6-alkylguanine-DNA alkyltransferase and DNase I activities in vitro by some alkylating substances and antineoplastic agents. 172 Jul 84

To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5'----3' exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.
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PMID:DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system. 221 24

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.
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PMID:Purification and characterization of primer recognition proteins from HeLa cells. 236 57

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.
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PMID:Complete enzymatic synthesis of DNA containing the SV40 origin of replication. 284 39

A DNA ligase has been purified approximately 2,100-fold, to near-homogeneity, from Drosophila melanogaster 6-12-h embryos and was shown to catalyze the formation of 3',5'-phosphodiester bonds. Polypeptides with molecular weights 83,000, 75,000, and 64,000 were observed when the purified enzyme was electrophoresed under denaturing conditions. These polypeptides were shown by partial proteolysis studies and two-dimensional gel analysis to be structurally related. The two smaller polypeptides were presumably derived from the largest, 83,000 molecular weight protein, by proteolysis during purification or in vivo. All three polypeptides formed enzyme-adenylylate complexes in the absence of DNA. Drosophila DNA ligase had a Stokes radius of 45 A, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.6, yielding a calculated molecular weight of 79,800. These studies indicate that DNA ligase from Drosophila embryos is a monomer. The purified ligase was free of detectable ATPase, nuclease, topoisomerase, and DNA polymerase activities. The enzyme exhibited an absolute requirement for ATP in the joining reaction. A divalent metal was required and N-ethylmaleimide inhibited the reaction. Formation of phosphodiester bonds by Drosophila ligase required the presence of 5'-phosphoryl and 3'-hydroxyl termini. The purified enzyme restored biological activity to endonucleolytically cleaved pBR322 DNA. The specific activity of Drosophila DNA ligase was highest in unfertilized eggs. Developing embryos had 5-10-fold more ligase activity than at any later time in development.
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PMID:DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization. 309 38

In the presence of AMP and Mg2+, a covalently closed duplex DNA containing negative superhelical turns was treated with DNA ligase isolated from bacteriophage T4-infected E. coli. This resulted in the gradual and not sudden loss of superhelical turns as for example in the case of type I DNA topoisomerase. All DNA products remain covalently closed. Since T4 enzyme-mediated DNA relaxation is inhibited by both pyrophosphate and by ATP this suggests that DNA relaxing and DNA joining activities probably coincide. EDTA addition in the presence of a large excess of enzyme, induces the formation of nicked DNA products while protein denaturing treatments are not very effective. Our observations might suggest an involvement of the relaxing activity of DNA ligase during the ligation process.
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PMID:AMP-dependent DNA relaxation catalyzed by DNA ligase occurs by a nicking-closing mechanism. 313 26

Administration of hepatocarcinogens aflataxin B1 (AFB1) and N-nitrosodimethylamine (NDMA) to rats caused single-strand breaks in hepatic nuclear DNA. The damage was found to be maximum at 4 hours following AFB1 administration and at 2 hours following NDMA administration. These damages were repaired after 17 and 4 hours, respectively in cases of AFB1 and NDMA. The activity of poly(ADP-ribose)polymerase (PARP), an enzyme known to use single-strand breaks of DNA as cofactor, was observed to increase with increasing damage to DNA and decrease as and when this damage got repaired. DNA polymerase beta and DNA ligase activities were also seen to increase and decline in a way analogous to PARP. In contrast, DNA topoisomerase activity declined corresponding to an increase in PARP activity. These observations suggest a possible role of PARP in coordinating the activities of other enzymes involved in DNA repair. It is also envisaged that these parameters can be utilized to devise strategies to counteract the deleterious effects of chemical carcinogens.
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PMID:Activity of some nuclear enzymes associated with DNA repair following hepatocarcinogen administration to rats. 759 30


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