Figure 4

Religation kinetics. (A) Gel analysis of the religation kinetics observed when incubating the wild type or the Glu710Gly mutant-suicide cleavage complex with the R11 complementary ligator oligonucleotide (shown at the top of the figure) in absence [lanes 3–6 and lanes 12–15 for wild type and Glu710Gly mutant respectively] or in presence of 100 μM CPT [lanes 7–10 for the wild type and lanes 16–19 for the Glu710Gly mutant]. In lane 1 no protein was added. The lanes 2 and 11 represent the time 0 for the wild type and the Glu710Gly mutant reactions, before the addition of the complementary R11 strand. “CL1” represents the DNA fragment cleaved at the preferred enzyme site; “religation” is the restored fully duplex oligonucleotide representing the final product of the religation reaction. (B) Plot of the percentage of disappearance of the cleavage complex relative to time 0, in absence or in presence of CPT for the wild type (triangles, full and dashed black lines, respectively) and the Glu710Gly mutant (squares full and dashed grey lines, respectively). Data shown are means ± SD from 3 independent experiments.