Binding site requirements for double-strand cleavage
Previous work with several zinc finger chimeric nucleases, including QQR, showed that they make cuts primarily to the left side of their recognition sequences, as depicted in Figure (22
). This was the expected location, given the orientation of the zinc fingers on the DNA and the structure of the chimeric protein. Some cleavage occurred on both strands, but the mapping of the sites was performed on denatured DNA and the efficiency of double-strand cleavage was not determined (22
). Therefore, we focused our attention on the production of double-strand breaks.
We constructed and analyzed a collection of specifically designed plasmid substrates with variable numbers and orientations of the canonical recognition site for QQR. These were linearized and treated with QQR. At enzyme:substrate ratios close to 1, in order to achieve double-strand cleavage it was necessary to have at least two copies of the target oligonucleotide (Fig. a). A single copy of the recognition sequence (pQS) did not support cleavage. With 10 bp between paired sites, both tail-to-tail inverted repeats (pQT10) and direct repeats (pQD10) were effectively cut, while head-to-head inverted repeats (pQH10) were cleaved much less efficiently. Observed double-strand breaks mapped to the expected sites (Fig. a and data not shown).
Figure 2 Substrate specificity of QQR. (a) Substrates with various binding site dispositions. pQS has a single copy of the canonical recognition site, indicated by the arrow. The remaining DNAs have two sites in tail-to-tail inverted (pQT10), head-to-head inverted (more ...)
At substantially higher enzyme:substrate ratios, both QQR and QNK made targeted cuts in DNAs that carried a single copy of the recognition site. In the comparison shown in Figure b, QS carries a single site, while QT16 has two in tail-to-tail orientation 16 bp apart. The DNAs were PCR fragments of ~200 bp, identical to those used for mapping reactions (see below). QT16 was cleaved at all QQR concentrations tested and cleavage was essentially complete at an approximately 1:1 ratio of enzyme to sites (lane 4). In contrast, QS required ~10-fold more enzyme to achieve comparable levels of cleavage (QS in lane 4 versus QT16 in lane 1), and this corresponds to a 20-fold higher ratio of enzyme to recognition sites. At the highest enzyme concentration used (lane 5), other sites began to be cleaved, perhaps reflecting binding of QQR to more distantly related sequences.
Influence of target site separation on cleavage efficiency
Paired inverted sites in the tail-to-tail orientation showed efficient double-strand cleavage when the sites were 10 or 16 bp apart (Fig. ). To determine the upper and lower limits on distances that would allow cleavage, we examined a series of substrates for each chimeric nuclease in which variable amounts of essentially random DNA sequence were inserted between the recognition sites. For QNK, separations of 8, 14, 28 and 48 bp were tested (Fig. a). Under conditions that did not support cleavage at a single site (pKS), the 8, 14 and 28 bp separations allowed double-strand cleavage, while the 48 bp separation did not.
For QQR, we tested a larger collection of different separations, as shown in Figure b. When the paired sites were 4 bp apart, very little double-strand cleavage was observed and that only at the highest enzyme input. A separation of 6 bp led to good cleavage with QQR and this remained true for all distances tested up to 35 bp. The substrate with a separation of 40 bp, however, was essentially not cleaved. Thus, the upper limit for effective site separations is between 35 and 40 bp, in agreement with the observations for QNK.
Mapping cut sites on DNA strands
In principle, the requirement for two binding sites to achieve double-strand cleavage could reflect either of two underlying phenomena. (i) Each individual bound chimeric molecule might make an independent single-strand cut close to its binding site and two such cuts in proximity would be necessary to produce a double-strand break. In this view the upper limit on the distance between effective paired sites would be determined by the stability of the DNA duplex between nicks on the two strands. (ii) The cleavage domain of the chimeric nuclease might have to dimerize in order to act as an effective nuclease and when it does concerted breaks would be made in the two strands. Natural Fok
I dimerizes to cleave DNA (25
) and it is reasonable to suspect that the cleavage domains in the context of the chimeric nuclease would do the same. In this case, the upper limit on effective site separation would reflect the maximum extension achievable by the peptide linker between the binding and cleavage domains.
We distinguished these possibilities by mapping the cut sites for QNK and QQR on a wide range of substrates at single nucleotide resolution. Model (i) predicts that single-strand cuts will be produced in fixed positions relative to each recognition site and that their locations will move apart as the distance between the sites is increased. Model (ii) predicts that cuts in the two strands will always be paired and, like FokI, they should produce a 5′ overhang of 4 bp.
To map the cuts made by QNK, a fragment carrying the paired sites, the intervening sequence and ~50 bp of pUC18 was labeled on either end with 32P as described in Materials and Methods. After digestion with the enzyme, products were compared to G and G+A sequencing reactions of the same fragment (Fig. a). Maxam–Gilbert chemistry removes the designated base and leaves the preceding 3′-phosphate, while the chimeric nuclease leaves a 3′-hydroxyl. Both these properties increase the mobility of the Maxam–Gilbert fragments, so the alignment with the QNK products was adjusted by about 1.5 bands to identify the exact site of cleavage.
Figure 4 Mapping cut sites on DNA strands. (a) QNK substrates. Lanes G and G+A contain Maxam–Gilbert sequencing reaction products of the end-labeled DNAs. Adjacent lanes have the same fragments (~40 nM) treated without enzyme (–) (more ...)
With 8 bp between QNK sites (KT8), strong cuts were seen on both strands between the sites: a single cut on one strand, a strong and a secondary cut on the other strand (Fig. a). When mapped on the DNA sequence, the major cuts are 4 bp apart and result in a 5′ overhang (Fig. a). With KT14, five or six relatively strong cuts were made on each strand (Figs a and a). When mapped they overlap considerably, but may be interpreted as three clusters of paired cuts staggered by ~4 bp, one near the middle of the intervening sequence and one near each end. With KT28, again a single strong cleavage site was seen on both strands near the middle of the space between binding sites with a 4 bp 5′ stagger. Minor bands were visible in all cases, indicating that the cut locations were not rigidly determined.
Cuts were also mapped on a DNA carrying a single recognition site for QNK (KS), using a high concentration of enzyme (Fig. a). Two groups of cuts were seen on each strand, similar to results obtained previously with other zinc finger chimeras (9
). These cuts assemble on the DNA sequence into two clusters centered ~4 and 13 bp from the 5′-end of the binding site (Fig. a). There is a general 5′ stagger in each cluster, although the distances between the cuts are not restricted to 4 bp. Similar locations were seen with KT48 at high QNK concentrations (Fig. a).
Also shown in Figure a are mapped cuts in two QNK substrates that were determined independently by the procedure described for QQR below. The major cuts in KT8′ are farther apart than seen in KT8, perhaps due to sequence preference of the FokI cleavage domain (see Discussion). KT12 showed paired, centered, strong cuts separated by a 4 bp 5′ stagger, plus one minor cut reflecting a 3 bp stagger.
To map cuts on QQR substrates, a PCR fragment of ~200 bp from each plasmid was labeled on either end and reaction products were analyzed in parallel with dideoxy sequencing reactions on the same DNAs, using the same primers. At moderate QQR concentration essentially no nicks were produced in the vicinity of a single copy of the recognition site (not shown). At the same concentration single strong cuts were made in both strands between sites separated by 12 bp (QT12). When mapped onto the DNA sequence, these strong cuts were precisely 4 bp apart with a 5′ stagger (Fig. b). With QT16, cuts were made near one end or the other of the intervening sequence and the most prominent cuts occurred in pairs with a 4 bp 5′ stagger. QT30 provided the only case in which the strongest cuts were clearly separated by <4 bp. These paired cuts staggered by 3 bp were located at the center of the spacer.
Examining the full range of QQR substrates in Figure b, we see that paired nicks were always located between the binding sites and related by a 5′ stagger of ~4 bp. With QT6, prominent cuts were made at the center of the interval with a 4 bp stagger. For QT8, the strongest cuts were centered and showed 5 or 6 bp staggers. QT10 showed alternative 4 bp staggers offset slightly from the center of the symmetrical intervening sequence and QT12 showed the centered 4 bp stagger described earlier. There were minor bands in each of these cases corresponding to slightly shorter or slightly longer 5′ staggers. With QT14, QT18 and QT20 the paired cuts on the two strands occurred not in the center of the intervening sequence, but close to either end, as described for QT16 above. While the major cuts were staggered by 4 bp in most cases, minor cut sites were also seen. In QT26, QT30 and QT35, the cut locations returned to the center of the interval, although weak cuts near the ends were seen with QT26. When the site separation reached 40 bp, no prominent nicks were seen, just as for the single site substrate.
These results support Model (ii), i.e. dimerization of the cleavage domain in the space between the binding sites.
Cleavage of paired non-identical sites
To achieve cleavage of an arbitrarily selected target, paired zinc finger binding sites would be chosen that would usually not be identical. To demonstrate that such a configuration also leads to effective dimerization and cleavage, we constructed a substrate having one site each for QNK and QQR 14 bp apart and in tail-to-tail inverted orientation (pQK14). This DNA was not cleaved by either enzyme alone at moderate enzyme concentration, but was cleaved by a mixture of the two (Fig. ). The level of cleavage was comparable to that of pQT14 using the corresponding QQR enzyme alone. Thus, paired non-identical sites are effective cleavage targets when enzymes with both specificities are provided.
The crystal structure of Fok
I reveals a dimer interface, where Asp483 of each cleavage domain monomer makes bidentate hydrogen bonds with Arg487 of the other (28
). Simultaneous conversion of these two residues to Ala dramatically reduced the cleavage efficiency of the restriction endonuclease (25
). In order to confirm the requirement for dimerization of the chimeric nucleases, we mutated each of these residues individually in QNK: Asp483 to Ala (D483A) and Arg487 to Asp (R487D). The corresponding proteins were purified and used to treat pKT8, which was readily cleaved by QNK. Both mutant enzymes were incapable of producing double-strand breaks (Fig. ). Thus, dimerization is critical for the activity of the chimeric nucleases, just as for native Fok