Heat-Induced DNA Relaxiation In Vitro by Mouse DNA Topoisomerase I in the Presence of Ethidium Bromide

In general, covalently-closed circular DNAs that are relaxed by DNA topoisomerase I at higher temperatures are more negatively (or less positively) supercoiled when analyzed at the same temperature. We found that in the presence of ethidium bromide the reaction products were less negatively supercoiled at higher temperatures. Consequently, the linking number of substrate DNA changed reversibly with temperature-shift: DNA relaxation occurred on temperature shift-up, and re-supercoiling on temperature shift-down. Analyses of the migration of covalently closed circular DNA on agarose gel electrophoresis and of the fluorescence intensity of ethidium bromide bound to DNA indicated decrease in binding of the drug to DNA at higher temperatures. Thus, the thermal effects on the topology of the reaction products of mouse DNA topoisomerase I in the presence of ethidium bromide can be explained by the temperature-sensitive interaction of the drug with DNA.