Topoisomerases are essential enzymes that modulate DNA topology by generating transient single- (type I enzymes) or double-stranded (type II enzymes) breaks in the double helix. Human cells encode a single form of topoisomerase I and two related topoisomerase II isoforms, alpha and beta. Beyond their essential physiological activities, topoisomerases are targets for several widely prescribed anticancer drugs. These agents kill cancer cells by increasing levels of transient topoisomerase-generated strand breaks, which in turn are converted to permanent DNA breaks by the movement of tracking systems (such as replication complexes) through topoisomerase-DNA cleavage complexes. Although the human genome is globally underwound (negatively supercoiled), DNA that immediately precedes tracking systems is overwound (positively supercoiled). Therefore, we examined the ability of human topoisomerases to cleave positively supercoiled DNA in the absence and presence of anticancer drugs. Positively supercoiled plasmids were prepared by treatment with reverse gyrase. Levels of topoisomerase IIalpha and beta-mediated DNA cleavage were ~3-fold lower with positively supercoiled molecules. Decreased scission also was observed with nonintercalative drugs such as etoposide. However, high DNA scission was seen with intercalative drugs such as amsacrine. This latter effect results from a decreased ability of intercalative agents to bind and further overwind positively supercoiled substrates. In contrast to topoisomerase II, topoisomerase I-mediated DNA cleavage was ~3-fold higher with positively supercoiled DNA. Furthermore, the enzyme was markedly more sensitive to the anticancer drug, camptothecin. Thus, the response of topoisomerases to anticancer drugs is strongly affected by DNA topology. Supported by NIH Grants GM33944 and CA09582.
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