Figure 3

(A) Kinetics of DNA relaxation by PAT. Lane 1 shows relaxed DNA. Supercoiled pUC18 (110 ng) was relaxed with 9 ng PAT in topoisomerase buffer. Lanes 2 through 10 contain 110 ng of DNA and 9 ng PAT. Reactions were stopped after 0.5, 1, 2, 3, 4, 5, 10, 15 and 20 minutes. The relaxed DNA (R) and supercoiled DNA (S) are identified on the left.(B) Relaxation of supercoiled pUC18 in the presence of PATCAT. Lane 1 shows the relaxed DNA. The supercoiled pUC18 (110 ng) was relaxed with 9 ng PAT i. Lanes 2 through 11 contain 110 ng DNA and 808.2 ng PATCAT. Reactions were stopped after 0.5, 1, 2, 3, 4, 5, 10, 15, 20, and 25 minutes in lanes 2 to 11 respectively. The relaxed and supercoiled DNA are identified by (R) and (S) on the left. (C) The Y292F mutant is not able to relax supercoiled pUC18. The relaxed and supercoiled DNA are identified by (R) and (S) on the left. In lane 1, is shown the negative control containing supercoiled pUC18 and lane 2 is showing the relaxed DNA (supercoiled pUC18 (110 ng) relaxed with PAT (9 ng)). Lanes 3 through 7 contain 110 ng DNA and 241 ng Y292F. Reactions were stopped after 5, 10, 15, 20 and 25 minutes, respectively.