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. 2006 May 18;25(11):2575–2583. doi: 10.1038/sj.emboj.7601142

Figure 4.

Figure 4

Relaxation kinetics of supercoiled chromatin. (A) Positively supercoiled Yp4.4 minichromosomes and their accompanying control plasmid pHC624 were incubated with catalytic amounts of topoisomerase I or topoisomerase II (as indicated). Reactions were quenched at the indicated periods (min). Following DNA electrophoresis, the gel-blots were probed for Yp4.4 (up) and for pHC624 (down). Note that topoisomerase I and II activities were adjusted to relax the control plasmid at comparable rates. (B) Mixtures containing (+) supercoiled Yp4.4 plasmid plus (−) supercoiled pHC624 plasmid, or containing (+) supercoiled Yp4.4 minichromosome plus (−) supercoiled pHC624 plasmid were relaxed with catalytic amounts of topoisomerase I or topoisomerase II (as indicated). Reactions were quenched at the indicated periods (min). DNA electrophoresis was carried at 25°C in a 0.9% agarose gel in TBE buffer at 40 V for 14 h. Gel-blots were probed for Yp4.4 plus pHC624. DNA relaxation rates, by topoisomerases I and II, for supercoiled minichromosomes (CHR S(+)) and supercoiled plasmids (DNA S(−), DNA S(+)) were determined by measuring the gain of relaxed topoisomers excluding nicked ones. Graphs represent the average of four experiments with error bars indicating s.d.'s from the mean.