Mechanistic studies of the modulation of cleavage activity of topoisomerase I by DNA adducts of mono- and bi-functional PtII complexes (original) (raw)
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Platinated DNA Adducts Enhance Poisoning of DNA Topoisomerase I by Camptothecin
Journal of Biological Chemistry, 2004
Camptothecins constitute a novel class of chemotherapeutics that selectively target DNA topoisomerase I (Top1) by reversibly stabilizing a covalent enzyme-DNA intermediate. This cytotoxic mechanism contrasts with that of platinum drugs, such as cisplatin, which induce inter-and intrastrand DNA adducts. In vitro combination studies using platinum drugs combined with Top1 poisons, such as topotecan, showed a schedule-dependent synergistic activity, with promising results in the clinic. However, whereas the molecular mechanism of these single agents may be relatively well understood, the mode of action of these chemotherapeutic agents in combination necessitates a more complete understanding. Indeed, we recently reported that a functional homologous recombination pathway is required for cisplatin and topotecan synergy yet represses the synergistic toxicity of 1--D-arabinofuranosyl cytidine in combination with topotecan (
Phenanthriplatin Acts As a Covalent Poison of Topoisomerase II Cleavage Complexes
ACS chemical biology, 2016
Drugs capable of trapping topoisomerase II (Top2), an essential enzyme that cleaves DNA to remove naturally occurring knots and tangles, can serve as potent anticancer agents. The monofunctional platinum agent phenanthriplatin, cis-[Pt(NH3)2(phenanthridine)Cl](NO3), is shown here to trap Top2 in addition to its known modes of inhibition of DNA and RNA polymerases. Its potency therefore combines diverse modes of action by which phenanthriplatin kills cancer cells. The observation that phenanthriplatin can act as a Top2 poison highlights opportunities to design nonclassical platinum anticancer agents with this novel mechanism of action. Such complexes have the potential to overcome current limitations with chemotherapy, such as resistance, and to provide treatment options for cancers that do not respond well to classical agents. Covalent DNA-platinum lesions implicated in Top2 poisoning are distinctive from those generated by known therapeutic topoisomerase poisons, which typically ex...
Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme
Biochimica Et Biophysica Acta-gene Structure and Expression, 1998
DNA topoisomerase I is essential for cellular metabolism and survival. It is also the target of a novel class of anticancer drugs active against previously refractory solid tumors, the camptothecins. The present review describes the topoisomerase I catalytic mechanisms with particular emphasis on the cleavage complex that represents the enzyme's catalytic intermediate and the site of action for camptothecins. Roles of topoisomerase I in DNA replication, transcription and recombination are also reviewed. Because of the importance of topoisomerase I as a chemotherapeutic target, we review the mechanisms of action of camptothecins and the other topoisomerase I inhibitors identified to date. 0167-4781 / 98 / $^see front matter ß
Induction of Topoisomerase I Cleavage Complexes by the Vinyl Chloride Adduct 1,N 6-Ethenoadenine
Journal of Biological Chemistry, 1998
We used purified mammalian topoisomerases I (top1) and oligonucleotides to study top1-mediated cleavage and religation in the presence of a potent carcinogenic adduct, 1,N 6-ethenoadenosine (⑀A) incorporated immediately downstream of a unique top1 cleavage site. We found tha ⑀A markedly enhanced top1 cleavage complexes when it was incorporated at the ؉1 position of the top1 cleavage. This enhancement was due to a reduction of the religation step of the top1 reaction. In addition, ⑀A reduced the top1-mediated cleavage and decreased binding of the enzyme to DNA. We also studied the effects of the ⑀A adduct on top1 trapping by camptothecin (CPT), a well known top1 inhibitor. CPT was inactive when ⑀A was present at the ؉1 position. Alkylation of the top1 cleavage complex by 7-chloromethyl-10,11-methylenedioxycamptothecin(7-ClMe-MDO-CPT) was also blocked by the ⑀A adduct. Altogether, these results demonstrate that the ⑀A carcinogenic adduct can efficiently trap human top1 and mimic CPT effects. Normal hydrogen bonding of the base pairs immediately downstream from the top1 cleavage site is probably essential for efficient DNA religation and binding of camptothecins in the top1 cleavage complex.
Biochimie, 1999
DNA topoisomerase II regulates the three-dimensional organisation of DNA and is the principal target of many important anticancer and antimicrobial agents. These drugs usually act on the DNA cleavage/religation steps of the catalytic cycle resulting in accumulation of covalent DNA-topoisomerase II complexes. We have studied the different steps of the catalytic cycle as a function of salt concentration, which is a classical way to evaluate the biochemical properties of proteins. The results show that the catalytic activity of topoisomerase II follows a bell-shaped curve with optimum between 100 and 225 mM KCl. No straightforward correlation exists between DNA binding and catalytic activity. The highest levels of drug-induced covalent DNA-topoisomerase II complexes are observed between 100 and 150 mM KCl. Remarkably, at salt concentrations between 150 mM and 225 mM KCl, topoisomerase II is converted into a drug-resistant form with greatly reduced levels of drug-induced DNA-topoisomerase II complexes. This is due to efficient religation rather than to absence of DNA cleavage as witnessed by relaxation of the supercoiled DNA substrate. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations. Unexpectedly, the addition of DNA stimulates ATP hydrolysis at 100 and 150 mM KCl, but has little or no effect below 100 mM KCl in spite of strong non-covalent DNA binding at these salt concentrations. Therefore, DNA-stimulated ATP hydrolysis appears to be associated with covalent rather than non-covalent binding of DNA to topoisomerase II. Taken together, the results suggest that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II providing a unifying theme for the biological and pharmacological modulation of this enzyme. © 1999 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS topoisomerase II / catalytic activity / topoisomerase inhibitors / covalent DNA-protein complexes / ATP hydrolysis
Journal of Biological Chemistry, 2004
Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr 727 of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp⅐DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn 726 with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn 726 with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.
Sequence specificity of DNA topoisomerase I in the presence and absence of camptothecin
The EMBO Journal, 1987
Previously, we have demonstrated that in Tetrahymena DNA topoisomerase I has a strong preference in situ for a hexadecameric sequence motif AZACTTAGAZAAATTA present in the non-transcribed spacers of r-chromatin. Here we characterize more extensively the interaction of purified topoisomerase I with specific hexadecameric sequences in cloned DNA. Treatment of topoisomerase I-DNA complexes with strong protein denaturants results in single strand breaks and covalent linkage of DNA to the 3' end of the broken strand. By mapping the position of the resulting nicks, we have analysed the sequence-specific interaction of topoisomerase I with the DNA. The experiments demonstrate that: (i) the enzyme cleaves specifically between the sixth and seventh bases in the hexadecameric sequence; (ii) a single base substitution in the recognition sequence may reduce the cleavage extent by 95%; (iii) the sequence specific cleavage is stimulated 8-fold by divalent cations; (iv) 30% of the DNA molecules are cleaved at the hexadecameric sequence while no other cleavages can be detected in the 1.6-kb fragment investigated; (v) the sequence specific cleavage is increased 2-to 3-fold in the presence of the antitumor drug camptothecin; (vi) at high concentrations of topoisomerase I, the cleavage pattern is altered by camptothecin; (vii) the equilibrium dissociation constant for interaction of topoisomerase I and the hexadecameric sequence can be estimated as-10-10 M.
Human topoisomerase I forms double cleavage complexes on natural DNA
Biochemical and Biophysical Research Communications, 2006
DNA topoisomerase I releases torsional stress generated in chromatin during transcription and replication. Usually topoisomerase I is recognized to work as a monomer, but previously we have shown that two molecules can form a dimer-like protein-protein complex on a 'suicide' DNA substrate resulting in a topoisomerase I double cleavage complex. Here we show that during the normal relaxation reaction a considerable fraction of human topoisomerase I formed transient dimers on plasmid DNA too. Recombinant as well as topoisomerase I purified from human cells formed double cleavage complexes within a distance of 12 or 14 nucleotides. When topoisomerase I was isolated from camptothecin-treated HeLa cells, a considerable fraction migrated to the same position as topoisomerase I bearing a covalently bound 12-to-14-mer oligonucleotide. Taken together our data suggest that human topoisomerase I double cleavage complexes are part of the normal catalytic cycle of this enzyme that occur in vitro and possibly also in vivo.
Modulation of relaxation activity of human topoisomerases by Pt(II)-based complexes
Journal of Inorganic Biochemistry, 2020
The clinical efficiency of Pt(II)-based drugs is founded on articulate mechanisms of action. Indeed it depends on a balanced combination of metal ion reactivity towards proteins and nucleic acids. Here we analysed the effect of two trans-platinum planar amines in comparison to cisplatin and transplatin on the DNA processivity by human topoisomerases I and IIα. Each tested metal complex produces DNA adducts with unique geometrical features and, consistently, they exert different effects on the activity of tested enzymes. Moreover, our results highlighted more subtle consequences on the enzymatic activity by the tested metal complexes which derive from a combination of preferential DNA or protein platination. Moreover, we observed that it is not possible to predict the overall output based only on the cis-vs trans-geometry of the tested metal complexes. This variable behaviour reflects the chemical reactivity profile of each single metal complex and can be usefully addressed to describe their different properties in the complex physiological environment.