A switch in the mechanism of communication between the two DNA-binding sites in the SfiI restriction endonuclease.

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.

Protein assembly and DNA looping by the FokI restriction endonuclease.

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein-protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.

Cleavage of individual DNA strands by the different subunits of the heterodimeric restriction endonuclease BbvCI.

BbvCI cleaves an asymmetric DNA sequence, 5'-CC downward arrow TCAGC-3'/5'-GC downward arrow TGAGG-3', as indicated. While many Type II restriction enzymes consist of identical subunits, BbvCI has two different subunits: R(1), which acts at GC downward arrow TGAGG; and R(2), which acts at CC downward arrow TCAGC. Some mutants of BbvCI with defects in one subunit, either R(1)(-)R(2)(+) or R(1)(+)R(2)(-), cleave only one strand, that attacked by the native subunit. In analytical ultracentrifugation at various concentrations of protein, wild-type and mutant BbvCI enzymes aggregated extensively, but are R(1)R(2) heterodimers at the concentrations used in DNA cleavage reactions. On a plasmid with one recognition site, wild-type BbvCI cleaved both strands before dissociating from the DNA, while the R(1)(-)R(2)(+) and R(1)(+)R(2)(-) mutants acted almost exclusively on their specified strands, albeit at relatively slow rates. During the wild-type reaction, the DNA is cleaved initially in one strand, mainly that targeted by the R(1) subunit. The other strand is then cleaved slowly by R(2) before the enzyme dissociates from the DNA. Hence, the nicked form accumulates as a transient intermediate. This behaviour differs from that of many other restriction enzymes, which cut both strands at equal rates. However, the activities of the R(1)(+) and R(2)(+) subunits in the wild-type enzyme can differ from their activities in the R(1)(+)R(2)(-) and R(1)(-)R(2)(+) mutants. Each active site in BbvCI therefore influences the other.

Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA.

Type IIs restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions, typically several base pairs away from the recognition site. These enzymes are generally monomers that transiently associate to form dimers to cleave both strands. Their reactions could involve bridging interactions between two copies of their recognition sequence. To examine this possibility, several type IIs enzymes were tested against substrates with either one or two target sites. Some of the enzymes cleaved the DNA with two target sites at the same rate as that with one site, but most cut their two-site substrate more rapidly than the one-site DNA. In some cases, the two sites were cut sequentially, at rates that were equal to each other but that exceeded the rate on the one-site DNA. In another case, the DNA with two sites was cleaved rapidly at one site, but the residual site was cleaved at a much slower rate. In a further example, the two sites were cleaved concertedly to give directly the final products cut at both sites. Many type IIs enzymes thus interact with two copies of their recognition sequence before cleaving DNA, although via several different mechanisms.