The Geometry of DNA Supercoils Modulates Topoisomerase-Mediated DNA Cleavage and Enzyme Response to Anticancer Drugs

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Abstract
Collisions with DNA tracking systems are critical for the conversion of transient topoisomerase−DNA cleavage complexes to permanent strand breaks. Since DNA is overwound ahead of tracking systems, cleavage complexes most likely to produce permanent strand breaks should be formed between topoisomerases and positively supercoiled molecules. Therefore, the ability of human topoisomerase IIα and IIβ and topoisomerase I to cleave positively supercoiled DNA was assessed in the absence or presence of anticancer drugs. Topoisomerase IIα and IIβ maintained ∼4-fold lower levels of cleavage complexes with positively rather than negatively supercoiled DNA. Topoisomerase IIα also displayed lower levels of cleavage with overwound substrates in the presence of nonintercalative drugs. Decreased drug efficacy was due primarily to a drop in baseline (i.e., nondrug) cleavage, rather than an altered interaction with the enzyme−DNA complex. Similar results were seen for topoisomerase IIβ, but the effects of DNA geometry on drug-induced scission were somewhat less pronounced. With both topoisomerase IIα and IIβ, intercalative drugs displayed greater relative cleavage enhancement with positively supercoiled DNA. This appeared to result from negative effects of high concentrations of intercalative agents on underwound DNA. In contrast to the type II enzymes, topoisomerase I maintained ∼3-fold higher levels of cleavage complexes with positively supercoiled substrates and displayed an even more dramatic increase in the presence of camptothecin. These findings suggest that the geometry of DNA supercoils has a profound influence on topoisomerase-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either topoisomerase IIα or IIβ.