Mechanism of the reaction catalyzed by the DNA untwisting enzyme: Attachment of the enzyme to 3′-terminus of the nicked DNA

Champoux, James J.

doi:10.1016/0022-2836(78)90238-3

Cited by 78 publications

(32 citation statements)

References 7 publications

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“…The mixture was incubated for 3 min at 37°C and then stopped by the addition of SDS to a final concentration of 1.5%. The topoisomerase in the reaction was precipitated by the addition of trichloroacetic acid to 10%, recovered by centrifugation, washed with acetone, and then resuspended in SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer (125 mM Tris [pH 6.8], 700 mM 2-mercaptoethanol, 20% glycerol, 4% SDS, 0.02% bromphenol blue), boiled, and analyzed by SDS-PAGE by using precast 7.5% acrylamide gels (Integrated Separation Systems). After electrophoresis, the gel was stained with Coomassie dye, dried, and autoradiographed with Kodak XAR ifim for 16 h.…”

Section: Methodsmentioning

confidence: 99%

Characterization of DNA topoisomerase I from Candida albicans as a target for drug discovery

Fostel

Montgomery

Shen

1992

Antimicrob Agents Chemother

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Candida albicans is an opportunistic pathogen responsible for life-threatening infections in persons with impaired immune systems. Topoisomerase I is a potential target for novel antifungal agents; however, in order for this enzyme to be a therapeutically useful target, it needs to be demonstrated that the fungal and human topoisomerases differ sufficiently as to allow the fungal topoisomerase to be selectively targeted. To address this question, we isolated the topoisomerase I from C. albicans and compared its biochemical properties with those of the mammalian enzyme. Similar to other eukaryotic type I topoisomerases, the C. albicans type I topoisomerase has an apparent molecular mass of 102 kDa and covalently links to the 3' end of DNA, as shown after the reaction is interrupted by sodium dodecyl sulfate. Topoisomerase poisons such as camptothecin act by stabilizing the cleavage complex formed by the topoisomerase I and DNA. We observed that the C. albicans and mammalian type I topoisomerases differ in that the C. albicans cleavage complex is approximately 10-fold less sensitive to camptothecin than the mammalian cleavage conmplex is. In addition, we found that the antifungal agent eupolauridine can stabilize the cleavage complex formed by both the C. albicans and human topoisomerases and that the response of the C. albicans topoisomerase I to this drug is greater than that of the human enzyme. Thus, the topoisomerase I from C. albicans is sufficiently distinct from the human enzyme as to allow differential chemical targeting and will therefore make a good target for antifungal drug discovery.Topoisomerases are enzymes which modulate the topological structure of DNA (for recent reviews, see references 7, 20, and 30). Topoisomerases are able to relax supercoiled DNA and unlink catenated DNA circles, among other functions. These enzymes perform these reactions by making a transient break in the DNA backbone, passing another segment of DNA through this break, and then resealing the break. Type I topoisomerases break only one DNA strand, while type II topoisomerases can introduce a double-strand break into DNA. In the eukaryotic cell, type I topoisomerases are associated with elongating transcription and replication forks, while type II topoisomerases are required for segregation of daughter chromosomes after cell division (1,12,13,18,34,37).Topoisomerases form a complex with DNA in which the transiently broken ends of the DNA are constrained. When a protein denaturant is added, a DNA break is revealed, and the denatured topoisomerase is found to be covalently attached to one end of the break (5,11,16,33,35). This complex between topoisomerase and DNA has been termed the cleavage complex (26; reviewed in reference 23). Certain drugs stabilize the cleavage complex, such as the anticancer drug camptothecin, which targets the eukaryotic topoisomerase I (19). A structure-activity relationship for camptothecin derivatives has been established, and a correlation is seen between the cytotoxicity of the compound in replic...

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Section: Methodsmentioning

confidence: 99%

Characterization of DNA topoisomerase I from Candida albicans as a target for drug discovery

Fostel

Montgomery

Shen

1992

Antimicrob Agents Chemother

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“…The anticancer drug 9-AC is a semisynthetic camptothecin analogue [1][2][3][4], an inhibitor of topoisomerase I, a nuclear enzyme implicated in the cellular processes of replication, transcription, recombination, DNA repair, and chromosome segregation [5][6][7][8]. Inhibition of topoisomerase I by 9-AC via stabilizing the complex formed by the enzyme and DNA results in arrest in the G2 cell cycle phase and cell death [9].…”

Section: Introductionmentioning

confidence: 99%

Biodistribution and pharmacokinetics of colon-specific HPMA copolymer–9-aminocamptothecin conjugate in mice

Gao

Lü

Kopečková

et al. 2007

Journal of Controlled Release

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A water soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer -9-aminocamptothecin (9-AC) conjugate was designed for oral colon-specific drug delivery for the treatment of colon cancer. Comparative studies between the polymer conjugate and free drug have been performed to assess their biodistribution and pharmacokinetics in mice. After oral administration of equal doses of the polymer conjugate or free 9-AC, the drug concentrations in major organs at fixed time points were determined using an HPLC-fluorescence assay. Only 2±1% of 9-AC released from the polymer conjugate was detected in small intestine (SI), and the mean peak concentration of free 9-AC was 45-fold higher than that from released drug. Colon-specific release of 9-AC produced high local concentrations. The mean peak concentration of released 9-AC in cecal contents, feces, cecal tissue, and colon tissue were, respectively, 3.2-fold, 3.5-fold, 2.2-fold and 1.6-fold higher than that using free 9-AC. In plasma, the high and sharp drug concentration profile from free drug was in contrast to the relatively low and flat pharmacokinetic profile obtained from drug released from the HPMA copolymer. There was no significant difference between released and free drug for the area under the concentration-time curve (AUC) and bioavailability values. As a consequence of the colonspecific release of unmodified 9-AC from the polymer conjugate, antitumor efficacy can be anticipated to be enhanced due to prolonged colon tumor exposure to higher and more localized drug concentrations.

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“…Topo-I introduces transient single-stranded DNA breaks in one of the phosphodiester backbones of the duplex DNA and results in a reversible Topo-I/DNA covalent complex (5)(6)(7). In 1999, we cloned and sequenced the first Topo-I gene from L. donovani (LdTOP1A) (4).…”

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confidence: 99%

Topoisomerase I Amino Acid Substitutions, Gly185Arg and Asp325Glu, Confer Camptothecin Resistance in Leishmania donovani

Marquis

Hardy

Olivier

2005

Antimicrob Agents Chemother

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The antitumor compound camptothecin (CPT) is also recognized for its specific activity against Leishmania donovani topoisomerase I (Topo-I). In consequence, defining CPT resistance mechanisms represents an important strategic tool in the acquisition of a better understanding of its mode of action. In the present study, we selected a single highly resistant L. donovani strain termed LdRCPT.160 by stepwise exposure to CPT. Gene sequencing revealed two single nucleotide mutations in the LdRCPT.160 LdTOP1A gene, resulting in two amino acid substitutions (Gly185Arg and Asp325Glu) in the protein. Moreover, these two substitutions observed in the LdTOP1A protein were correlated with a decreased Topo-I DNA relaxation activity in these resistant parasites. Nevertheless, there was no change in the LdTOP1A gene expression level. Interestingly, transfection studies of the LdRCPT.160 LdTOP1A gene in its wild-type counterpart showed that it induced CPT resistance. Sitedirected mutagenesis studies demonstrated that, despite a substantial level of resistance conferred by the Gly185Arg and Asp325Glu substitutions separately, both were essential to reach a high-resistance phenotype. Of interest, the amino acid substitutions observed in LdRCPT.160 LdTOP1A protein occurred near the amino acids previously predicted to interact with CPT, providing new insight into the mechanism of CPT molecular action.

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Mechanism of the reaction catalyzed by the DNA untwisting enzyme: Attachment of the enzyme to 3′-terminus of the nicked DNA

Cited by 78 publications

References 7 publications

Characterization of DNA topoisomerase I from Candida albicans as a target for drug discovery

Characterization of DNA topoisomerase I from Candida albicans as a target for drug discovery

Biodistribution and pharmacokinetics of colon-specific HPMA copolymer–9-aminocamptothecin conjugate in mice

Topoisomerase I Amino Acid Substitutions, Gly185Arg and Asp325Glu, Confer Camptothecin Resistance in Leishmania donovani

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