DNA restriction dependent on two recognition sites: activities of the Sfi I restriction–modification system in Escherichia coli

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Abstract
In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4–6 bp long, the Sfi I endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the Sfi I endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the Sfi I restriction–modification system were transformed with plasmids carrying Sfi I sites. The Sfi I system often failed to restrict the survival of a plasmid with one Sfi I site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated Sfi I sites were not restricted. No rearrangements of the plasmids carrying Sfi I sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, Sfi I displays all of the hallmarks of a restriction–modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro. For both restriction in vivo and DNA cleavage in vitro, Sfi I operates best with two recognition sites on the same DNA.