Cleavage/Religation Equilibrium.

(a) Gel electrophoresis of the products generated by incubation of hTop1 (lanes 1-8) and hTop1(pf-Linker) (lanes 9-16) with the [γ -³²P] end-labelled duplex DNA (CL25/CP25), shown at the top of the figure. The arrow indicates the preferred cleavage site. The duplex was incubated for different time intervals with hTop1 in absence (lanes 1-4) and in presence of CPT (lanes 4-8) or with hTop1(pf-Linker) in absence (lanes 9-12) and in presence of CPT (lanes 13-16). Lane 17, no enzyme added. The band corresponding to the enzyme–substrate cleaved complex, has been indicated by an asterisk. (b) Percentages of the hTop1 (black) or hTop1(pf-Linker) (grey) cleavage complex generated after 1 minute of incubation normalized to the total amount of radiolabeled DNA in each lane. Data shown are means ± SD from 3 independent experiments.