4780 Nucleic Acids Research, 2005, Vol. 33, No. 15 speciﬁc DNA sequence. Homodimeric forms of Mod.EcoPI and Mod.EcoP15I (Mod2) have been shown to act independ- ently as methyltransferases, both recognizing and modifying their target sequences independent of Res (30,31). The Mod.PstII sequence shares amino acid features with the methyltransferase regions of Mod.EcoP15I and Mod.EcoPI ( 30% identity, 46% similarity, data not shown). The pstIIR overlaps pstIIM by 4 nt. A similar arrangement is seen in other Type III systems (1), although mod and res genes may actually be expressed from separate promoters rather than as a poly- cistronic transcript (32). Res.PstII has amino acid motifs [Figure 1, (28)] and predicted secondary structural features (33) characteristic of both an endonuclease (Region X) and a superfamily 2 DNA helicase. The Type III helicase motifs can be classiﬁed on the basis of sequence alignments and predicted secondary structures in one of two groups, Type IIIA and Type IIIB (33). Whilst Res.EcoP15 and Res.EcoPI are Type IIIA, Res.PstII falls into the Type IIIB group (align- ments are shown for helicase Motif I in Figure 1B). The recognition site of PstII To ascertain the recognition sequence of PstII we chose to use an in vitro approach (Materials and Methods). Our ﬁrst step was to purify the complete RM system from E.coli TOP10 cells transformed with one of the PstII clones (Materials and Methods, Figure 1C). The ﬁnal puriﬁed samples contained a Res:Mod ratio of 1:3. In the accompanying paper (15), we show that maximum PstII endonuclease activity is likely to be obtained with a Res2Mod2 assembly as shown for EcoPI/ EcoP15I (34). However, in contrast to the E.coli complexes, assembly of the PstII heterotetramer is dynamic and the reac- tion conditions used in this paper (Materials and Methods) were chosen to favour saturation of the DNA sites with the Res2Mod2 form rather than sub-assemblies that have reduced endonuclease activity and enhanced methyltransferase activity (15). All concentrations of the PstII mixture were quoted in terms of a Res:Mod ratio of 1:3. We observed that unregu- lated expression of the genes from the native promoters on a high copy number plasmid resulted in low transformation efﬁciencies and that some transformants contained inactive protein (data not shown). It would appear that horizontal trans- fer of PstII can result in cell death (most likely due to DNA breakdown). This is similar to observations with StyLTI (8) but is in contrast to the more freely-transferable EcoPI and EcoP15I genes [(35) and references therein]. In these latter cases restriction activity is delayed until methylation is com- plete as Mod regulates the expression of Res with additional control at the translational level. Mapping using both DNA cleavage and methylation activ- ities identiﬁed the recognition sequence of PstII as CTGATG (Materials and Methods, Figure 2A). This analysis relied partly on the assumption that PstII endonuclease activity requires two indirectly-repeated copies of its recognition site as shown for other Type III enzymes such as EcoPI and EcoP15I (36,37). For instance, for an EcoP15I substrate to be cleaved it must carry a 50-CAGCAG-30 sequence (Figure 2A) followed by a 50-CTGCTG-30 sequence on the same strand—any other arrangement is incompatible with cleavage expect under certain special conditions. To conﬁrm that this assumption is true for PstII we measured cleavage activity on DNA substrates with different numbers of CTGATG sites (Figure 2B); either zero sites [pSKFokI, (38)], one site [pAT153, (18)], two directly-repeated sites (pLJP11a) or two indirectly-repeated sites (pLJP11b). The only substrate that could be cleaved in the presence of 4 mM ATP was pLJP11b (Figure 2C). No cleavage was seen with 4 mM ATPg S (data not shown). The presence or otherwise of 100 mM AdoMet did not affect the outcome of the reactions under these conditions. The ﬁnal product of the reaction was the FLL DNA cut at one or other site (the cleavage loci were mapped precisely as described below, Figure 3). No further Figure 3. Identification of the PstII cleavage loci using four sites with different flanking sequences. (A) Sequences of sites 1 and 2 from pLJP11b (Figure 2B), site 3 from pLJP12c and site 4 from pLJP12d. PstII recognition sequence in bold, shared flanking sequences underlined. Cleavage loci are indicated by an arrow. (B) Representative denaturing PAGE (site 2) showing separation of primer extension products following PstII cleavage. See main text for full details. A 32P-labelled DNA generated by single round primer extension was cut with PstII to give a labelled product as shown (lines represent DNA, arrowhead represents PstII site as in Figure 2A). Sample ‘ ’ was then run directly on the gel, whilst sample ‘+’ was first treated with Klenow polymerase. Sizes of the resulting fragments were compared to sequencing reactions produced using the same primer. For presentation purposes the brightness and contrast of the digital images were differentially adjusted using a linear intensity scale to increase the relative contrast of the labelled fragments.