Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

Abstract

Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting 'top' and 'bottom' strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting.

Figure 1.

Experimental strategy. In all three panels, both strands of the DNA duplex are shown, with gaps to indicate cleaved products: the yellow box marks the recognition site for FokI. The FokI monomer is shown as two domains connected by a flexible linker: a (large) DNA recognition domain for specific binding and a (small) catalytic domain for dimerization and DNA cleavage. Functional domains are in blue. Domains inactivated by mutation for either DNA binding (in the large domain) or for catalysis (in the small domain) are in red. The 1° monomer bound to the recognition site could in principle use its catalytic domain to engage the scissile bond in the top strand, leaving the 2° monomer to attack the bottom strand (pathway i in all three panels). Alternatively (pathway ii), the 1° monomer attacks the scissile bond in the bottom strand, in which case the 2° monomer cuts the top strand. (A) With two monomers of wt FokI, both pathways (i) and (ii) lead to the cutting of both strands. (B) With a mixture of wt FokI and the N13Y-D450A double mutant, only the wt enzyme can act as the 1° monomer, while either the wt or the double mutant might act as the 2° monomer. Hence, the double mutant ought to inhibit the reaction on the strand cut by the 2° monomer: the bottom strand in pathway (i); the top in pathway (ii). (C) With the D450A and the N13Y mutants, D450A can bind to the specific site but N13Y cannot. As only N13Y is active, D450A and N13Y have to function as 1° and 2° monomers, respectively so this mixture ought to cut only one strand: the bottom strand in pathway (i); the top in (ii).

Figure 2.

Strand selection by wt FokI. (A) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42, ³²P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S_t or S_b) and cleaved strands (P₁₆ or P₁₂) marked on the left. (B) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. (C) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min⁻¹ and 0.3 min⁻¹ for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min⁻¹ and 0.5 min⁻¹ for top and bottom strands, respectively.

Figure 3.

Strand-specific nicking. (A) A mixture of the N13Y and D450A mutants of the FokI endonuclease was added to immobilized BIO-42 to give a reaction with 10 nM N13Y, 10 nM D450A and 1 nM DNA in Buffer 4 at 20°C. The BIO-42 was ³²P-labelled in either the top (left-hand gel) or the bottom strand (right-hand). At various times after adding the mixture, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown. The electrophoretic mobilities of the intact strands (S_t and S_b, from top- and bottom-strand labelled BIO-42, respectively) are noted on the left of the gels and the lanes marked Wt show the products from reactions of wt FokI on the same DNA species (P₁₆ from the top strand, P₁₂ from the bottom). (B) The fraction of the total amount of radiolabel in each lane still present as the intact DNA were measured and these values plotted as a function of reaction time: top strand, black circles; bottom strand, white circles. Error bars denote standard deviations from ≥3 independent repeats. The line drawn through the data from the top strand (solid line) is the best fit to a single exponential, which gave a rate constant of 0.3 min⁻¹. Data points for the bottom strand are connected by a dashed line.

Figure 4.

Specificity of nicking. The reactions, in Buffer 4, contained 5 nM SC pSKFokI (a plasmid with one recognition site for FokI) and FokI protein as indicated below. Reactions were stopped after 1 h at 37°C and the samples analysed by electrophoresis through agarose. The symbols SC, OC and LIN on the right of each gel mark the electrophoretic mobilities of the intact SC DNA, the nicked OC form cut in one strand and the LIN form cut in both strands at one site. The lanes marked M contain 1 kb electrophoresis markers (NEB), and the lanes marked Wt are from equivalent 1 h reactions of 10 nM wt FokI on pSKFokI. (A) Left-hand gel: the reactions contained D450A at the concentrations indicated above each lane (0 → 200 nM). In the right-hand gel, the reactions with 10 → 200 nM D450A also contained 10 nM N13Y. (B) As (A) except that the protein whose concentration was varied was N13Y and that, in the right-hand gel, the samples with varied N13Y also contained 10 nM D450A.

Figure 5.

Reaction rates. The reactions, in Buffer 4 at 37°C, contained 5 nM ³H-labelled DNA and FokI protein at either 1 nM or 10 nM, as indicated below. At timed intervals after adding the protein(s) to the DNA, samples were removed, quenched and analysed as described in Materials and methods section to obtain the concentrations of the SC, OC and LIN DNA. The residual concentration of SC DNA at each time point is given as a percentage of the total DNA in that sample. (A) Reactions of 1 nM wt FokI on: pSKFokI (one recognition site), black circles; pIF190 (two FokI sites), white circles. (B) Reactions with a mixture of N13Y and D450A, both at 1 nM, on pSKFokI (black circles) and on pIF190 (white circles). Also shown in (B) are the reactions with the mix of N13Y and D450A, both at 10 nM, on pSKFokI (black squares) and on pIF190 (white squares), both with dashed lines.