New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site.
A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 mM, while the religation reaction seemed unaffected by concentrations as high as 3 M-NaCl. The divalent cations Mg2+, Ca2+ and Mn2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu2+ and Zn2+ inhibited both reactions. Furthermore, the effect of the anti-neoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation.
Svejstrup
J Q
JQ
Department of Molecular Biology and Plant Physiology, University of Aarhus, Denmark.
Christiansen
K
K
Gromova
I I
II
Andersen
A H
AH
Westergaard
O
O
D000970:Antineoplastic Agents / Q000494:pharmacology; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000187:drug effects / Q000378:metabolism*; D004277:DNA, Single-Stranded; D005057:Eukaryotic Cells / Q000201:enzymology*; D006801:Humans; D012965:Sodium Chloride
D000970:Antineoplastic Agents; D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D012965:Sodium Chloride; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin