Fig. 2. Comparison of the cleavage activities by Int and its C-domain. (A) Topoisomerase activities of λ Int and the C-domain. Supercoiled pBR322 plasmid DNA (0.36 µg/assay) was incubated with the indicated amounts of protein at 25°C for 1 h (see Materials and methods). The reactions were quenched in 0.2% SDS and analyzed by agarose gel electrophoresis. The positions of supercoiled (s.c.) and relaxed (rel.) plasmid DNA comprise the two ends of the ladder of topoisomers. (B) DNA cleavage as a function of protein concentration. ³²P-labeled top-strand nicked full att-site suicide substrate (0.02 µM) was incubated with the indicated amounts of Int or C-domain for 30 min at 25°C and reactions were quenched with 0.2% SDS. (C) DNA cleavage as a function of time. Int or C-domain (0.1 µM) was incubated for the indicated times and reactions were quenched in 0.2% SDS. In both (B) and (C), the reaction conditions were as described in Materials and methods and the amount of covalent complex formed was analyzed by SDS–PAGE and quantitated using a phosphorimager (Fuji).