Analysis of topoisomerase I-mediated reorganization of DNA structure

The relative abilities of eleven camptothecin derivatives to enhance calf thymus topoisomerase I-mediated cleavage of synthetic DNA duplex have revealed that the A ring of camptothecin is very important for its biochemical activity. Depending on the type, number, and location of substituents, highly active or inactive analogues were obtained. The persistence of CPT-induced calf thymus topoisomerase I-DNA covalent binary complexes was investigated by using substrates DNA containing one or several topoisomerase I cleavage sites. The dissociation rates of the individual CPT derivatives at a given topoisomerase I cleavage site, following NaCl treatment, were sometimes quite different and not directly correlated with their efficiencies in stabilizing enzyme-DNA binary complex. Individual camptothecin derivatives also exhibited a spectrum of inhibitory potencies in preventing the calf thymus topoisomerase I-mediated rearrangement of branched, nicked, and gapped DNA substrates. These observations demonstrated the complicated interactions of the CPTs with varied binary complexes formed between topoisomerase I and DNA.; Recombinant human topoisomerase I was expressed in a baculovirus system and purified to apparent homogeneity by FPLC heparin chromatography. The human topoisomerase I was shown to mediate the rearrangement of branched, nicked, and gapped DNA substrates with different rates and efficiencies than that of the calf thymus enzyme. Additionally, several of the CPT analogues exhibited substantially different effects on DNA structure transformations mediated by calf thymus and human topoisomerases I. The role of the 20-OH group in camptothecin binding by the human topoisomerase I-DNA binary complex was investigated by using five 20-substituted CPT analogues. Both 20-chloro- and 20-bromo camptothecin bound as well to the enzyme-DNA binary complex as 20-amino CPT. These results suggested that the two proposed CPT binding models require further refinement. Also described is the significantly different pH rate profiles of recombinant human topoisomerase I-mediated strand cleavage and religation of oligonucleotide DNA under single turnover conditions, indicating possible differences in the rate-limiting steps in individual enzymatic processes.