A Switch in the Mechanism of Communication between the Two DNA-Binding Sites in the SfiI Restriction Endonuclease

Abstract

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.

Fidelity of DNA Sequence Recognition by the SfiI Restriction Endonuclease Is Determined by Communications between Its Two DNA-Binding Sites

“…18,24 Y68F at this concentration would have cleaved to completion a two-site substrate in ∼30 min and a one-site substrate in b3 min. 21 When increasing concentrations of either wt SfiI or Y68F were added to pUC19, a plasmid that has one star site but no true site for SfiI, neither enzyme cleaved the plasmid to any detectable level (Fig. 1a).…”

“…18 SfiI recognises an interrupted 8 bp palindrome, GGCCNNNN↓NGGCC (where ↓ marks the point of cleavage and N is any base 19 ) but has low activity when bound to a single site. 20,21 It shows its maximal activity when bound simultaneously to two copies of this sequence: this occurs most readily with two sites in cis, on the same molecule of DNA, when it loops out the intervening DNA. 22,23 SfiI then normally cuts both strands at both sites before dissociating from the DNA, although conditions that lessen DNA-protein affinities such as elevated salt concentrations lead to the release of products cut at only one site or in just one strand.…”

“…27 The removal of this hydroxyl group, by replacing the tyrosine with phenylalanine to give the Y68F mutant, severely perturbed the communication between the two DNA-binding clefts. 21 The Y68F protein retained the tetrameric structure of wild-type (wt) SfiI and its capacity to bind two DNA duplexes concurrently. On DNA substrates with two SfiI sites, Y68F displayed a lower turnover rate than the wt enzyme, due to a slower release of the cleaved DNA products, but it was faster than the wt on substrates with one target site.…”

“…Hence, while wt SfiI cleaves two-site DNA more rapidly than one-site DNA, 18,22,24 the exact converse holds for the Y68F mutant: it cleaves DNA with one SfiI site faster than DNA with two sites. 21 Indeed, when bound to a single specific duplex that had been tethered to preclude any possibility of interactions with two duplexes in trans, 17 the Y68F protein cleaved the duplex more rapidly than did the wt enzyme. 21 Consequently, if the reason why wt SfiI needs to interact with two copies of its recognition sequence is to act as a double-check safety mechanism against DNA cleavage events at inappropriate sequences, then the ability of the Y68F protein to act at solitary DNA sites ought to be accompanied by a lower discrimination against non-cognate sequences.…”

“…Wt SfiI has a relatively low activity [Plasmids and enzymes were purified as before. 16,18,21 Typically, about 90% of the plasmid preparations were the supercoiled (SC) monomeric form, with b 10% nicked open circle (OC) DNA or the dimeric form generated by homologous recombination; the dimer (not indicated) runs just below the OC DNA in the agarose gels]. After 1 h at 50°C, 10 μl of stop mix was added and the samples analysed by electrophoresis through agarose: 15,22,24 the electrophoretic mobilities of the SC, OC and full-length linear (LIN) forms of the DNA are marked on the right, as are also the products from reactions involving at least one star site (⁎).…”