Figure 6

(A) Relaxation of supercoiled DNA in the presence of different cations. Reactions contained 200 ng of supercoiled pUC18 and the indicated cation at 2.5 mM final concentration in topoisomerase buffer without MgCl₂ and 0.24 M NaCl. Lane 1: relaxed DNA (R); relaxation of 200 ng supercoiled pUC18 was performed with 9 ng of PAT in topoisomerase buffer with MgCl₂ for 2 hours at 37°C. Lane 2 contains 200 ng supercoiled pUC18 in the topoisomerase buffer indicated by (S) on the left hand side. Lanes 3 through 6 contain 9 ng of PAT. (B) Reactions contain 75 ng of supercoiled pUC18, 9 ng PAT, indicated cation at 2.5 mM concentration. The relaxed DNA (R) and supercoiled pUC18 (S) are identified on the left and were obtained as described in (A). Control reactions with metals only and no enzyme reveal no nicking activity (data not shown). (C) Effect of NaCl on topoisomerase activity. Reaction mixtures contained 110 ng of supercoiled pUC18 DNA, 2.5 mM EDTA, 9 ng PAT, and increasing concentration of NaCl 265 mM, 290 mM, 315 mM, 340 mM, 365 mM, 390 mM, 415 mM, 440 mM, 465 mM, 490 mM, 540 mM and 590 mM in lanes 3 through 14 respectively in topoisomerase buffer without MgCl₂. Lane 1 shows relaxed DNA and lane 2 is the supercoiled pUC18 DNA. The relaxed DNA (R) and supercoiled pUC18 (S) are identified on the left and were obtained as described in (A). Gels in panels (A) and (C) were electrophoresed at a higher voltage, reducing the separation between relaxed and supercoiled species.