4784 Nucleic Acids Research, 2005, Vol. 33, No. 15 occurred, the CCC substrate was always converted directly to FLL product with no build-up of the OC intermediate (full proﬁles not shown). The rate of rNTP hydrolysis was measured at a range of concentrations using a continuous coupled assay (Materials and Methods). No activity was observed in the absence of polynucleotide (data not shown). As DNA substrates we util- ized either a zero-site plasmid pSKFok1, (38) or a one-site plasmid pAT153, (18). Using 2-site DNA did not produce more than a linear increase in Type III NTPase activity (data not shown)—there is no apparent cooperativity when multiple sites are present. The rate on the speciﬁc DNA (NTP hydro- lysed per DNA site per min) was corrected for the substantial background rate on the non-speciﬁc DNA (Figure 6D). As elsewhere in this paper we utilized a high concentration of the PstII mixture (129 nM) to favour formation of the tetramer (15). This signiﬁcant excess of enzyme over sites is likely to be the cause of the high background rate. Combined with the low levels of ATPase activity observed, this resulted in corres- pondingly high levels of experimental deviation such that some apparent negative rates were recorded (this is where the background rate was higher that the speciﬁc rate). It should also be noted that due to limitations of the experimental set-up, rates could only be collected over tens of minutes whereas cleavage would have ﬁnished in tens of seconds (Figure 6C). Therefore, it is not clear how the observed rates relate to the true NTP consumption preceding a cleavage event. Neverthe- less, the apparent NTPase rates in Figure 6D show a pattern similar to those in Figure 6B and C in that ATP hydrolysis was favoured over CTP or GTP hydrolysis. The relatively slow NTPase rates observed (Figure 6D) are similar in magnitude to the ATPase rates measured previously with EcoPI and EcoP15I (47,48). Very little UTP hydrolysis could be meas- ured. However, by titrating increasing concentrations of UTP (0–8 mM) against a ﬁxed concentration of ATP (4 mM), an inhibition of the cleavage reaction was observed (Figure 6E). This suggests that UTP can compete with ATP for the nuc- leotide binding site but that UTP cannot be coupled efﬁciently to hydrolysis or DNA cleavage. The NTPase kinetics observed did not clearly match a Michaelis–Menten relationship (data not shown). The true relationship is being investigated in more detailed studies (A. Sears and M. D. Szczelkun, unpublished data). Non-Michaelis–Menton kinetics have also been observed with EcoPI (48). Long-distance interactions between enzymes The data in this paper shows that PstII must communicate between a pair of distant sites in head-to-head orientation in order to cleave a DNA substrate. It has been suggested for the Type I RM enzymes that DNA cleavage can be initiated at any site where a translocating enzyme stalls (52,53). A speciﬁc protein–protein interaction may not be required as two unrelated Type I enzymes can mutually activate cleav- age of a linear DNA which is not cut by either enzyme alone (52). Similarly, an interaction between EcoPI and EcoP15I has been shown to result in mutual activation of cleavage (34,54). However, the EcoPI and EcoP15I proteins are so similar in sequence as to be considered, fundamentally, as the same enzyme. Therefore, it is not clear if the com- munication between Type III tetramers require a speciﬁc Figure 7. The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. (A) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (l) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A. (B) Cleavage of l and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of l or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 mM. Following incubation for 1 h at 37 C, substrate and products were separated by agarose gel electrophoresis. protein–protein interaction or if a simple stall in translocation is sufﬁcient. To address the communication question we tested if the less closely related PstII and EcoP15I enzymes could interact to cleave T7 phage DNA (Figure 7). It has been noted previously that T7 phage DNA is refractory for cleavage by EcoP15I as all 36 recognition sites are in the same directly-repeated ori- entation (37,47,55,56). Strand bias is also true of the 34 PstII sites [Table 1, (55)] and, accordingly, T7 DNA is refractory to cleavage by PstII (Figure 7). The PstII and EcoP15I recogni- tion sites share a common sequence motif, CANCAG. Signi- ﬁcant strand bias is also observed in the other two versions of this motif (Table 1). The possible signiﬁcance of this obser- vation is discussed more in detail below. Using the deﬁnition of Type III site directionality as shown in Figure 2A, the PstII and EcoP15I sites in T7 can be said to be in a head-to-head orientation (Figure 7A). We utilized this fortuitous arrange- ment to test if a mixture of the two could interact and mutually activate cleavage. Lambda phage DNA in which strand bias of PstII and EcoP15I sites is not observed was utilized as a control (Figure 7A). On T7 DNA, neither PstII alone nor EcoP15I alone gener- ated any double-strand breaks, even after extensive incubation (Figure 7B, lanes 8–10). Combining the two enzymes in the same reaction did not alter this result (lanes 11–12) suggesting that EcoP15I and PstII cannot interact to cut both strands of a DNA substrate. This differs from the situation observed with Type I enzymes (54) or with EcoP15I and EcoPI (37,56). On lambda phage DNA both PstII and EcoP15I produced a ladder of product fragments resulting from cleavage at multiple sites