DNA restriction dependent on two recognition sites: activities of the Sfilrestriction-modification system in Escherichia coli

In contrast to many type II restriction enzymes, dimeric proteins that clea ve DNA at individual recognition sites 4-6 bp long, the Sill endonuclease i s a tetrameric protein that binds to two copies of an elongated sequence be fore cutting the DNA at both sites. The mode of action of the Sill endonucl ease thus seems more appropriate for DNA rearrangements than for restrictio n. To elucidate its biological function, strains of Escherichia coil expres sing the Sill restriction-modification system were transformed with plasmid s carrying SfiI sites. The SfiI system often failed to restrict the surviva l of a plasmid with one Sill site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated SfiI sites were not restricted. No rearrangements of the plasmids carrying Sill sites were det ected among the transformants. Hence, provided the target DNA contains at l east two recognition sites, Sfi I displays all of the hallmarks of a restri ction-modification system as opposed to a recombination system in E. coil c ells. The properties of the system in vivo match those of the enzyme in vit ro. For both restriction in vivo and DNA cleavage in vitro, SfiI operates b est with two recognition sites on the same DNA.