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Proteins that act at specific DNA sequences bind DNA randomly and then …
A series of 2.7-kb plasmids were constructed with two BbvCI sites separated by varied distances, from 30 to 75 bp. (Because the products from cutting the plasmids at one or both sites could not be separated from each other by electrophoresis, the reactions were studied on 301-bp fragments with both sites, generated by PCR across the relevant section of each plasmid.) To minimize local sequence effects, both sites had the same flanking sequences for 5 bp around the sites. For each construct, two derivatives were isolated: one with sites oriented in direct (head-to-tail) repeat so that one strand carries both copies of the CC sequence and the other both copies of GC, the targets for R2 and R1, respectively (Fig. 1a); another with sites in inverted (head-to-head) orientation so that the two CC sequences are in opposite strands, and likewise GC (Fig. 1b).
For processive action, BbvCI must translocate from one site to the other without leaving the domain of the DNA. With directly repeated sites, this translocation could occur by the protein sliding from one site to the other. If both subunits maintain contact with the DNA, the R2 subunit remains in place to cleave its target phosphodiester bond in the CC strand and likewise the R1 subunit the GC strand (Fig. 1a). However, processive action by BbvCI on a DNA with inverted sites cannot, for the reasons given below, arise solely from sliding and must include a dissociation/reassociation step. Consequently, if the translocation from one site to the other occurs solely by sliding, processivity will be observed with repeated sites but not with inverted sites. If it occurs mainly by sliding, processivity will be observed on both repeated and inverted sites although at a higher level on the repeated sites. However, if the translocation always involves a dissociation step, the extent of processivity on repeated sites will equal that on inverted sites.
If the BbvCI enzyme stays in contact with the DNA as it translocates between inverted sites, both its R1 and its R2 subunits face at the second site not only the “wrong” strand but also the wrong phosphodiester bond (Fig. 1b). To act processively on inverted sites, the protein must rotate around the helical axis of the DNA, to switch the R2 and the R1 subunits to the opposite strands, but it also must turn itself over from left to right along the DNA, to switch the active sites of both subunits onto the target bonds in their respective strands. For example, after BbvCI has cleaved the left-hand site in Fig. 1b and translocated to the right-hand site, a simple rotation of the R2 subunit around the long axis of the DNA will place that subunit onto its cognate CC strand, although in the wrong polarity. Its active site then will be opposite the second phosphodiester bond from the left, the ApG step in the bottom strand. However, to cleave the inverted sequence, the active site of R2 must engage the fifth phosphodiester bond in the bottom strand, the CpT step.
The rotation around the DNA could conceivably occur while the protein remains bound to it. However, the left-to-right turnaround along the DNA cannot occur while bound to DNA, because it requires the transmutation of protein through DNA, unless the DNA-binding surface of the protein is a flat or a convex plane with no protrusions. If the DNA-binding surface is a flat plane, then it may be possible to turn the DNA around from left to right while keeping it parallel to the plane, but it is physically impossible to turn the DNA around at any angle to the plane. However, protein surfaces are seldom, if ever, smooth planes with no protrusions. If the DNA binds in a concave depression in the protein surface, or if part of the protein protrudes into or around the DNA, then the rotation in the plane of the DNA-binding surface is blocked by steric hindrance. For example, if the DNA binds in a cleft within the protein structure, as is the case with the restriction enzymes bound to specific or nonspecific DNA (5), it may be possible to rotate the protein around the helical axis of the DNA following the screw axis of the helix (23), but it cannot be rotated around any other axis without first taking the DNA completely out of the cleft. Even if the protein just protrudes into the DNA grooves, as is usually the case with DNA-binding proteins (1), the rotation of the DNA around any axis other than its long axis is still blocked. The protein thus can reorient itself relative to the second site only when it is physically separated from the DNA by a sufficient distance to allow for the turnover without encountering a steric block from the protrusions on the surface of the protein and/or the DNA.
After the physical separation of protein from DNA, the protein will reassociate more often with the same DNA than with a different molecule (8, 11). Directly after the dissociation of a DNA–protein complex, the protein may remain near that DNA for some time while sampling all possible orientations relative to the DNA (13). This situation arises because a protein at a particular position in an aqueous solution will, as a result of diffusion, tumble relatively rapidly around that position but only rarely undergo lateral motion to a new position (6–8). Hence, even though the action of BbvCI on a DNA with two inverted sites requires its dissociation from the DNA before cutting the second site, it still has the potential to act processively on such a DNA. Extrapolation of data from the EcoRV nuclease (14, 25) suggests that, after leaving its recognition site on a 301-bp DNA (as here), it may reassociate with the same DNA 10–100 times before escaping to bulk solution.
In the substrates made here, the distances between the BbvCI sites are defined by the number of bp separating the target bonds in the CC sequences rather than the GC sequences. For substrates with repeated sites, the scissile bonds in the CC and the GC strands are the same distance apart (Fig. 1a). For sites in inverted (head-to-head) orientation, the GC → GC separation targeted by the R1 subunit is 6 bp shorter than the CC → CC span for the R2 subunit (Fig. 1b). The GC → GC distance is, however, immaterial, for the following reasons. First, the assays described below measure products with double-strand breaks, and the rate at which wild-type BbvCI cuts both strands is limited by R2 on the CC strand, not by R1 on the GC strand (31). Second, some experiments were done with DNA that had been nicked at both sites in the GC sequence by using the mutant of BbvCI (30). The nicked DNA was tested with wild-type BbvCI, which used its subunit to complete the double-strand break. The levels of processivity on the nicked DNA, which requires only the CC → CC transfer, were similar to those on the intact DNA, which require both CC → CC and GC → GC transfers (data not shown).
BbvCI Reactions. On a linear DNA with two sites, the first hit may be a distributive reaction at site 1 alone, to yield fragments A and BC, or at site 2 alone, to give AB and C (Fig. 2a). If these are the only possibilities, the initial rate for forming A (vA) must equal that for BC (vBC); likewise, vAB and vC (19, 25). [In addition, vBC will match vAB if the sites are equally susceptible, but not otherwise (19). Because the sites are both near the middle of the DNA (Fig. 2a) and are flanked by identical sequences, they should be equal. The similarity of vBC and vAB (namely, Fig. 2c) show that they are indeed equal.] After the initial reactions, the species carrying a BbvCI site, BC and AB, can be cleaved in subsequent reactions to yield their component fragments, but these reactions have no effect on the initial rates of product formation. However, instead of distributive reactions at single sites, the first hit may be a processive reaction to yield directly the three final fragments by cutting both before leaving the DNA. If so, the yield of BC will fall below that of A (as in Fig. 2c): likewise, AB relative to C. The proportion of processive reactions, relative to the total number of reactions, can be calculated from
Reactions on 301-bp substrates with two BbvCI sites, in either repeated or inverted orientation, were carried out under steady-state conditions with a 100-fold molar excess of DNA over enzyme (Fig. 2). At this DNA:enzyme ratio, virtually no DNA molecules bind two molecules of enzyme at the same time, so the direct cleavage of the DNA at both sites can only be due to processivity and not to separate events at solitary sites. Samples from the reactions were analyzed by electrophoresis through nondenaturing polyacrylamide under conditions that separate the substrate and all five of the possible reaction products from each other (Fig. 2b). [The DNA with inverted sites 75 bp apart yields a central product (B) of 72 bp that is seen on the gel (Fig. 2b). The substrates with shorter intersite spacing sites gave central products that migrated off the gel.] The DNA was labeled with 32P, and the amounts of the substrate and products in each sample were quantified (Fig. 2c). To ensure that the reaction velocities correspond to initial rates, only data from the first 15% of DNA cleavage were used.
Length and Salt Dependencies. Four pairs of substrates were tested. In one, the two BbvCI sites were separated by 30 bp; in the others, they were separated by 40, 45, and 75 bp. In each pair, one substrate had sites in direct repeat; the other had sites in inverted orientation. All four pairs were examined under four sets of conditions: without NaCl and with 30, 60, or 150 mM NaCl. The concentration of MgCl2 was kept constant at 5 mM; experiments at 10 mM MgCl2 gave much lower fP values (data not shown). In all cases, the initial velocities, vA, vBC, vAB, and vC, were measured as in Fig. 2, and the values were converted to processivity factors (Table 1). For each intersite spacing and reaction condition, the fP value across repeated sites (R) was compared with that on inverted sites (I) by taking the ratio, R/I (Fig. 3).
On the DNA with two sites 30 bp apart, BbvCI displayed in the absence of NaCl a high level of processivity on the substrate with repeated sites but a lower level on the isogenic substrate with inverted sites: the fP on inverted sites was ≈70% of that on repeated sites (Table 1). If the enzyme cuts one site on a linear DNA with two sites and then leaves that site by sliding onto the adjacent DNA with no directional preference, the maximal fP is 50%, because only 50% of the enzyme molecules head toward the intact site (19). In the absence of NaCl, the processivity observed on the DNA with repeated sites 30 bp apart, 46%, is close to the maximum. The levels of processivity on both substrates with sites 30 bp apart fell progressively as the NaCl concentration was raised, but processivity was still detected at 150 mM salt: ≈15% of the reactions were then processive. Strikingly, as the salt was raised, the difference between repeated and inverted sites declined, and no difference in fP values was detected in 150 mM NaCl. The R/I ratio thus fell from a value of 1.4 in the absence of NaCl to 1.0 in the presence of 150 mM NaCl, with intermediate values at 30 and 60 mM NaCl (Fig. 3).
Increasing the length of DNA between the sites from 30 to 40 or 45 bp, distances that approximate to 1 or 1.5 extra helical turns, had little effect on the fP values (Table 1). As with the 30-bp separation, the DNA molecules with sites 40 or 45 bp apart gave high levels of processivity in the absence of NaCl and progressively reduced levels at higher ionic strengths. Once again, they gave higher fP values on repeated sites than on inverted sites at low salt but very similar values for the two orientations at higher salt concentrations. Their R/I ratios thus declined as the salt was raised, from ≈1.3 in the absence of NaCl to 1.0 in the presence of 150 mM NaCl (Fig. 3).
The two substrates with 75 bp between the sites also were cleaved with high processivity in reactions lacking salt and with progressively lower levels of processivity at elevated ionic strengths (Table 1). However, in marked contrast to the shorter separations, the DNA with repeated sites 75 bp apart gave, at all NaCl concentrations tested, fP values that were virtually identical to those on the equivalent DNA with inverted sites (Table 1). Even in the absence of NaCl, the R/I ratio from the two substrates with sites 75 bp apart was close to 1.0; it had been 1.4 for the DNA with 30 bp between the sites (Fig. 3).
At 30 and 60 mM NaCl, the fP values for repeated sites 75 bp apart were similar to those for inverted sites 40 or 45 bp apart and were smaller than those on repeated sites 40 or 45 bp apart (Table 1). BbvCI thus translocates with similar efficiencies between two inverted sites 40–45 bp apart as it does between either repeated or inverted sites 75 bp apart. Presumably, these processes all proceed by the same pathway, whereas the higher efficiency observed on repeated sites 40–45 bp apart indicates an additional pathway.
The levels of processivity on both repeated and inverted sites declined as the salt was raised (Table 1). To see whether this result was due to salt reducing the affinity of BbvCI for nonspecific DNA, the binding of BbvCI to a 21-bp duplex that lacked the recognition sequence was examined by gel-retardation. The binding buffers were the same as the reaction buffers but with CaCl2 as a noncatalytic cofactor in place of MgCl2. BbvCI bound to the nonspecific duplex with KD values of ≈100 nM in either zero or 30 mM NaCl, ≈200 nM in 60 mM NaCl, and ≈500 nM in 150 mM NaCl (data not shown). The decline in processivity thus can be assigned to the effect of salt on weakening nonspecific binding. The affinities and the salt dependency of nonspecific DNA binding by BbvCI are comparable with other restriction enzymes (34–37).
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