Figure 1.

EcoR124I cleaves two DNA substrates sequentially. Representative agarose gels from sequential cleavage assays are presented. Reactions were done at 37°C and contained 4.4 nM active EcoR124I holoenzyme and 5 nM DNA (2.9 kB) in phase I. To initiate phase II, 5 nM pPB455 (R⁺-DNA; 9.8 kB) was added at t = 10 min. Following reaction completion, samples were subjected to electrophoresis in agarose gels as described in Materials and Methods section. In (A), the first DNA substrate was R⁺-DNA and in (B), the first substrate was 2R⁺-DNA. The position of migration of each band was compared to molecular weight markers present in the same gel (data not shown).