Kent Christiansen

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.

8-Methoxycaffeine inhibition of Drosophila DNA topoisomerase II

We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase II and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug.

Stimulation of Human DNA Topoisomerase I By Protein Kinase C

DNA topoisomerases are ubiquitous enzymes which exert important functions in replication, transcription, and chromatid segregation through regulation of DNA topology (Wang, 1985). Recently, topoisomerase I has also been identified as the primary cellular target for the antitumor drug, camptothecin, and derivatives thereof (Andoh et al, 1987, Zhang et al., 1990). Regulation of topoisomerase I activity might therefore represent one possible mechanism of controlling cellular activity and proliferation as well as directing the cytotoxic effects of clinical relevant antitumor drugs. Topoisomerase I relaxes DNA by concerted single strand cleavage and religation of the DNA backbone (Wang, 1985). The cellular level of mammalian topoisomerase I remains constant throughout the cell cycle and during different proliferative stages, whereas the activity of the enzyme has been found to vary (Hwang et al., 1989), suggesting regulation at the posttranslational level. Thus, the enzyme activity can in vitro be modified by poly-(ADP-ribosylation) and phosphorylation (Kasid et al., 1989, Samuels et al, 1989, Pommier et al, 1990). Here, we report that protein kinase C stimulates human topoisomerase I in vitro and describe the effect on the enzymatic activity in the absence and presence of camptothecin. Finally, we present a new method for purification of catalytic active forms of topoisomerase I from whole cell extracts.