4782 Nucleic Acids Research, 2005, Vol. 33, No. 15 adenine residue on one strand (Figure 2A), in a similar manner to other Type III enzymes (42,43). The PstII experiments illustrates a number of key points: (i) DNA cleavage requires a pair of recognition sites in cis on the same DNA molecule; (ii) the two sites must be arranged ‘head-to-head’ such that the sites appear on one strand as 50-CTGATG-30 followed by 50-CATCAG-30; (iii) hydrolys- able ATP, but not AdoMet, is absolutely required for cleavage; (iv) only one of the two sites is ever cleaved; (v) DNA cleav- age occurs 25–26 bp downstream of the CTGATG strand and generates a 2 bp 50-overhang; (vi) methylation of the site occurs on only one strand at a speciﬁc adenine residue. These are precisely the results predicted by the general Type III model. We also demonstrate in the accompanying paper (18) that DNA cleavage only results when a stoichiometric or greater amount of protein is added suggesting that, as with other Type III enzymes, PstII cannot turnover once it has cut the DNA. We tested a number of other buffers (both K+- and Na+-ion based) but did not observe any differences in the cleavage characteristics (data not shown). We did not observe any secondary (2 ) cleavage events. 2 events occur with EcoPI and EcoP15I in the presence of excess enzyme under relaxed buffer conditions, in particular the absence of the speciﬁcity factor AdoMet (36). It may be that because of the weak association of the Res and Mod subunits in PstII (15), we cannot add sufﬁcient tetrameric enzyme to our in vitro experiments to activate a 2 event. DNA cleavage activity The rate of DNA cleavage of the supercoiled 2-site substrate pLJP11b was analysed (Figure 5). The reactions were initiated by adding an excess of PstII to DNA sites to favour formation of Res2Mod2 complexes (Materials and Methods). Although this may result in a slow second order binding step preceding the cleavage kinetics, we have adopted this approach in Figure 5. Rate of DNA cleavage by the PstII restriction endonuclease. pLJP11b (5 nM) was incubated for various times with saturating PstII and 4 mM ATP at 37 C (see main text for full details). Where indicated AdoMet was included at 100 mM. The CCC substrate (squares) was separated from the OC intermediate (triangles) and FLL product (circles) by agarose gel electrophoresis (data not shown). Fragments were excised and the percentages of the 3H-labelled DNA fragments quantified by scintillation counting. general with Type III enzymes (36,44) to avoid co- methylation inhibiting the reaction before we can start to measure cleavage. The rate of digestion of pLJP11b was rapid compared to other Type III (and Type I) restriction enzymes (36,45) with the majority of the substrate cleaved in 10 s at 37 C. AdoMet did not affect this rate (the rate of appearance of FLL DNA is compared in Figure 5). Even at temperatures down to 4 C, PstII had relatively high endonuc- lease activity (data not shown). At all temperatures the inter- mediates nicked in one or other strand did not accumulate signiﬁcantly during the reaction and the CCC substrate was converted directly to the FLL product. Similar proﬁles have been observed with EcoP15I and EcoPI (36,39,46). Given that Type III enzymes only work under ‘single turnover’ conditions ([protein] > [DNA sites]), these proﬁles can be explained by one of two simple models, either: (i) that the rate for cleaving the ﬁrst strand is slower than that for cleaving the second; or, (ii) that there is a rate-limiting step prior to cleavage of the ﬁrst strand. We did not observe any differences in the proﬁles when reactions were carried out using Na+-based buffers (data not shown). NTPase activity Given the Type IIIB helicase motifs in the Res.PstII subunit and the ATP-dependent DNA cleavage shown in Figures 2 and 5, it was expected that PstII would have an ATPase activity equivalent to those measured previously for other Type III enzymes (47,48). We ﬁrst investigated the ability of a range of nucleotide triphosphates to activate DNA cleavage by PstII and EcoP15I (Figure 6A). A total of 5 nM pLJP11b (for PstII) or pMDS34a (for EcoP15I, 44) were incubated for 1 h at 37 C with excess enzyme and 4 mM nucleotide, as indicated. In addition to ATP, DNA cleavage by PstII was also activated by dATP, ddATP, GTP, dGTP, CTP and dCTP. Incubation with ddGTP produced some OC intermediate (but no FLL product) whilst ddCTP and all forms of UTP/TTP failed to activate cleavage. In comparison, EcoP15I was only activated by aden- osine triphosphates (Figure 6A) as observed previously (49). Some 2 cleavage of the DNA was observed with EcoP15I (these are relaxed buffer conditions). As above, no secondary cleavage was observed with PstII. Although hydrolysis of a full range of NTPs, dNTPs and ddNTPs has been observed for other DexH- and DEAD-box helicases (50), to the best of our knowledge this is the ﬁrst report of a Type III (or Type I) restriction enzyme that can utilise nucleotides other than those based on adenosine. Given the similarities in the ATP binding afﬁnities of PstII and EcoP15I (see below), we do not believe that ATP contamination can explain why PstII is active with GTP or CTP when EcoP15I is not. Hydrolysis of the ddNTPs is difﬁcult to measure using our coupled assay (Materials and Methods) because the coupling enzymes become rate-limited. Since ATPgS does not facilitate cleavage, we do not believe that binding alone by dNTPs or ddNTPs is sufﬁcient but, instead, that hydrolysis must occur. The dependence of the cleavage reaction on the relative concentrations of the NTPs was examined by measuring the quantity of FLL DNA liberated following cleavage for 1 h at 37 C (Figure 6B). DNA cleavage was complete at ATP con- centrations above 3 mM whilst two-orders of magnitude more CTP or GTP was required, with a slight preference