(lanes 1–6). Addition of AdoMet resulted in some inhibition due to co-methylation (lanes 4–6). However, combining EcoP15I and PstII actually generated more cleavage than either enzyme alone (compare lanes 2 and 3 to lane 5)— this is due to the combined cleavage of two independently acting systems. Therefore the inactivity on T7 cannot be due to non-speciﬁc inhibition of either PstII or EcoP15I by the partner enzyme. Instead, DNA cleavage by Type III enzymes must require a very speciﬁc interaction between the same (or closely related) proteins. DISCUSSION PstII acts as a prototypical Type III enzyme As part of a general screening programme to search for new restriction modiﬁcation systems, a segment of genomic DNA from P.stuartii 164 was cloned which carried a putative Type III RM system which has been named PstII [Results, Materials and Methods, (1)]. Our initial characterization of the enzyme activity of PstII indicated that it conformed to the prototypical Type III RM model as exempliﬁed by EcoPI and EcoP15I. In fact, the recognition and cleavage sites of PstII show remark- able similarities to the EcoP15I sites (Figure 2A and Table 2). The differences in the Type IIIA and IIIB Res motifs [Figure 1, (33)] therefore, do not result in gross changes in enzyme mechanism. We did observe a relaxed speciﬁcity in the nuc- leotide hydrolysis of PstII that would reﬂect a looser arrange- ment of residues in the NTP binding site. Since the helicase motifs cluster in this pocket (33,57), it is possible that other Type IIIB enzymes will also utilize NTPs other than ATP. In the accompanying paper (15) we describe how disassembly of PstII by loss of a Res subunit can act to control relative modiﬁcation/restriction activities. This appears to differ from EcoPI/EcoP15I where the tetramer is stable at nanomolar con- centrations in vitro (34). It is possible that this difference may reﬂect alternative routes to prevent autorestriction by Type III enzymes [see Discussion (15)]. We did note that PstII was apparently not as freely transferable as EcoPI or EcoP15I suggesting that despite very similar enzyme activities, the control of those activities is different in vivo. As we have observed previously for other Type III enzymes (36), AdoMet is not strictly required for DNA cleavage by PstII. Co-factor contamination was estimated as <15% with our preparations of PstII (data not shown), so it cannot be argued that every enzyme already has AdoMet bound. Instead Table 2. Survey of strand bias of CANCAG sequences in representative bac- teriophage genomes CTGCTG (e.g. EcoP15I) 50–30 30–50 CTGATG CTGGTG CTGTTG (e.g. PstII) 50–30 30–50 50–30 30–50 50–30 30–50 Phage l 41 T7 36 FA1122 42 T5 65 T3 48 T4 57 P1 34 P2 17 31 0 1 45 6 58 32 25 43 34 29 29 30 50 37 41 22 0 0 35 0 86 10 17 15 35 32 40 30 41 67 10 9 1 4 28 5 47 59 12 12 0 1 38 4 36 28 10 11 23 20 38 15 46 33 11 Nucleic Acids Research, 2005, Vol. 33, No. 15 4785 we conclude that it is a general feature of Type III enzymes that, under certain conditions, AdoMet is not required as an allosteric activiator. We did not observe any 2 cleavage events in the absence of the AdoMet. This may be because AdoMet does not have a ‘speciﬁcity factor’ role in PstII but perhaps a more likely explanation is that we could not add sufﬁcient excess enzyme to induce the additional cleavage events. Given the dynamic assembly of PstII (15), this is a difﬁcult question to pursue further. How does PstII communicate between DNA distant sites? In the standard Type III model (2–4,47), communication is believed to be due to enzyme complexes bound at each site hydrolysing ATP and translocating towards each other along the intervening DNA—cleavage is activated when the enzymes collide. If this model is correct, and assuming enzyme collision occurs on average halfway between the sites, each PstII complex on pLJP11b would need to travel 528 bp (one-half of the length of the DNA arc between the head-to-head sites, Figure 2B). With a maximum ATPase rate of 40 ATP/site/min (Figure 6D), and given that production of the ﬁrst strand break occurs within 10 s (Figure 5), it would appear that only 6–7 ATP molecules will have been con- sumed by each PstII tetramer before cleavage is initiated. This corresponds to an apparent coupling of 82 bp translocated for each ATP hydrolysed. This ‘step size’ is signiﬁcantly larger than anything else measured for a DNA helicase or ATP-dependent DNA translocase (58–62). Either the Type III enzymes utilize their helicase motifs to move on DNA in a completely new and highly efﬁcient manner, or they do not translocate at all. In comparison to the Type I RM enzymes, our knowledge of the motor mechanism of the Type III enzymes is somewhat murky (33). There is clear evidence that simple DNA looping does not play a role (44). However, there has yet to be a direct measurement of a 1D linear translocation activity. It is proven that the Type I enzymes extrude a DNA loop during translo- cation [(13) and references therein] and on the basis of the shared SF2 helicase motifs, this model has also been adopted for the Type III RM system (47): there is evidence both for (63) and against (34,44,64) loop extrusion by Type III enzymes. In this study we have shown for the ﬁrst time that the Type III communication requires speciﬁc interactions between distant enzymes (Figure 7). The glaring problem we now need to address is precisely how the Type III enzymes couple relatively low ATPase rates to communication events between distant DNA sites. The PstII enzyme will provide a valuable test system to compare with the EcoPI and EcoP15I enzymes. Strand bias in phage DNA as a marker of Type III activity A striking feature of the PstII recognition sequence is its similarity to that of the EcoP15I enzyme (Figure 2A): the sites share a common sequence motif, CANCAG. Although the Mod subunits of these enzymes have evolved to recognize a similar sequence, the assembly of the protein complex onto that site has not placed the catalytic residues at same relative positions—the locations of the methylated adenines and non-speciﬁc cleavage loci are quite different (Figure 2A). Given that both sequences show a strand bias in T7 phage