The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence.

The SfiI endonuclease cleaves DNA by a mechanism that differs from other restriction enzymes. While most restriction enzymes are dimeric proteins that interact with a single DNA site, SfiI exists in solution as a tetramer and it appears to interact with two copies of its recognition sequence before it can cleave DNA. Its primary reaction is then to cut both strands at both sites in a concerted process. The two sites can be on either the same or different DNA molecules, so SfiI provides a test system for long-range interactions on DNA. On either supercoiled or linear DNA with two sites separated by 1 kb, the bridging interaction between the sites is an intramolecular event: the majority of the DNA is converted directly to products cleaved at both sites, bypassing intermediates cut at one site. Sites on separate DNA molecules, or two sites on linear DNA several kb apart, engage in an intermolecular interaction prior to cleavage. The interaction between two DNA molecules with one site on each is impeded by supercoiling in both partners but is permitted when one partner is linear: it may require reptation of one DNA through another. SfiI reactions have marked similarities to some of the reactions catalysed by site-specific recombination enzymes.