The SfiI endonuclease is a prototype for DNA looping. It binds two copies of its recognition sequence and, if Mg2þ is present, cuts both concertedly. Looping was examined here on supercoiled and relaxed forms of a 5.5 kb plasmid with three SfiI sites: sites 1 and 2 were separated by 0.4 kb, and sites 2 and 3 by 2.0 kb. SfiI converted this plasmid directly to the products cut at all three sites, though DNA species cleaved at one or two sites were formed transiently during a burst phase. The burst revealed three sets of doubly cut products, corresponding to the three possible pairings of sites. The equilibrium distribution between the different loops was evalu-ated from the burst phases of reactions initiated by adding MgCl2 to SfiI bound to the plasmid. The short loop was favored over the longer loops, particularly on supercoiled DNA. The relative rates for loop capture were assessed after adding SfiI to solutions containing the plasmid and MgCl2. On both supercoiled and relaxed DNA, the rate of loop capture across 0.4 kb was only marginally faster than over 2.0 kb or 2.4 kb. The relative strengths and rates of looping were compared to computer simu-lations of conformational fluctuations in DNA. The simulations concurred broadly with the experimental data, though they predicted that increasing site separations should cause a shallower decline in the equilibrium con-stants than was observed but a slightly steeper decline in the rates for loop capture. Possible reasons for these discrepancies are discussed
