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. 2000 Nov 15;14(22):2881–2892. doi: 10.1101/gad.838900

Figure 2.

Figure 2

Kinetics of relaxation of (−) and (+) supercoiled DNA by topo IV. (A) Negatively supercoiled 2.7-kb DNA (ς = −0.05) was reacted at a molar ratio of 0.2 topo IV/DNA, and samples were removed at one-min intervals. Relaxation was analyzed by electrophoresis through an agarose gel containing 1 μg/mL chloroquine. An autoradiograph of a Southern blot of the gel is shown. (B) Quantification of relaxation of the (−) SC substrate at molar ratios of 0.1 (○) and 0.2 (●) topo IV/DNA. The rates of relaxation were one strand passage/enzyme/min at both stoichiometries. Negative supercoils were removed at a lower stoichiometry than in Fig. 1, because the DNA concentration was fivefold higher. (C) Positively SC 2.7-kb DNA (ς ∼ +0.05) was reacted with topo IV at a stoichiometry of 0.0025 and samples were removed at 30-sec intervals. Relaxation was analyzed by agarose gel electrophoresis. An autoradiograph of a Southern blot of the gel is shown. The positions of nicked DNA (N), relaxed topoisomers (R), and (+) supercoiled DNA (SC) are indicated. (D) Quantification of the data for relaxation of the (+) supercoiled substrate at stoichiometries of 0.0025 (○) and 0.005 (●). The rates of relaxation were 19 and 29 strand passages/enzyme/min at stoichiometries of 0.0025 and 0.005, respectively.