cleavage was observed at the remaining intact site. On a cir- cular DNA the indirectly-repeated sites can be classiﬁed as being in both ‘head-to-head’ and ‘tail-to-tail’ orientations [the deﬁnition of the ‘direction’ of the site (Figure 2A) is due in part to the location of the cleavage loci, see below]. To distinguish between these arrangements we measured cleavage activity on two linear versions of pLJP11b in which the sites are in either head-to-head (linHtH) or tail-to-tail (linTtT) arrangement (Figure 2B). Of these, only linHtH was cleaved (Figure 2C) at either site 1 (to produce 1822 and 2445 bp fragments) or site 2 (to produce 1390 and 2877 bp fragments); the 1055 bp fragment that would be released if both sites were cut was never observed. A preference for site 1 over site 2 was observed (in the ratio 68:32, data not shown) which is likely to be due to differences in the ﬂanking non-speciﬁc DNA sequences (Figure 3). Similar preferences in both cleavage and methylation have been observed for other Type III enzymes using sites with dissimilar ﬂanking sequences (6,39). When ﬂanking sequences are identical, cleavage is divided equally between the two sites [e.g. for EcoPI, (36)]. Another assumption of the mapping experiments was that DNA cleavage occurred at non-speciﬁc sites immediately adjacent to the recognition site (Materials and Methods). To characterize the cleavage sites more in detail we mapped the PstII strand breaks using the sequencing approach of Janulaitis et al. (40). In brief, the site 2 plasmid substrates pLJP11b, pLJP12c and pLJP12d were subjected to primer extension in the presence of [a-32P]ATP to generate dsDNA with one labelled strand. Each primer was extended over the DNA arc containing the sites in head-to-head orientation so that the labelled DNA was a substrate for PstII (i.e. equivalent to linHtH in Figure 2C). This DNA was cut with PstII and the sample split into two aliquots which were analysed by denaturing PAGE; one aliquot was loaded directly onto the gel whilst the other was ﬁrst treated with Klenow polymerase in the presence of excess dNTPs (Materials and Methods). Where the ‘top’ (50–30) strand is labelled, the ﬁrst sample gives the location of the cleavage site on the top strand. The length of the second sample relative to the ﬁrst then reports on the cleavage of the unlabelled bottom (30–50) strand: if cleavage generates a 50-overhang, then the top strand will be extended; if cleavage generates a 30-overhang, then the top strand will be digested back; if cleavage generates a blunt end, then no change in length will be observed. We analysed four PstII sequences that differed in their ﬂanking sequences (Figure 3A): sites 1 and 2 from pLJP11b, site 3 from pLJP12c and site 4 from pLJP12d (pLJP12c and pLJP12d also carry site 1 which was not examined). Cleavage of the top strand mapped to non-speciﬁc loci 25–26 bp 30-to the recognition site. In every case treatment with Klenow polymerase generated a fragment extended by 2 nt indicating a 50-overhang 2 nt in length. These cleavage loci and their relative locations are similar to those observed for EcoP15I [(41), Figure 1A]. The 1 nt difference in spacing of the cleav- age site at site 1 compared to the other sequences is likely to be due to DNA structural features local to the scissile phospho- diester bonds (39) and/or the exact number of base pairs per helical turn in the intervening DNA. As well as the Type III enzymes, some Type IIS and IIB enzymes that also cut non- speciﬁc DNA adjacent to their sites have been shown to cleave imprecisely (1,40). Nucleic Acids Research, 2005, Vol. 33, No. 15 4781 Figure 4. Identification of the PstII methylation site. (A) Oligonucleotides were synthesized and annealed to generate non-specific and specific DNA substrates. Pre-modified sequences were generated by synthesizing oligonu- cleotides with an N6 methyl deoxyadenosine at either position 1, 2 or 3. (B) Scintillation counting of oligonucleotide substrates following incubation with PstII and [3H-methyl]-AdoMet. See main text for full details. To pinpoint the recognition site residue(s) methylated, we measured the modiﬁcation of a series of synthetic oligonuc- 3 leotide substrates using [ H-methyl] AdoMet (Materials and Methods, Figure 4). Janulaitis et al. (40) have used a similar procedure with Type II enzymes, e.g. AloI. Each dsDNA substrate was treated with PstII for 3 h in the presence of [3H-methyl] AdoMet and in the absence of NTPs. Under these conditions methylation will occur without any DNA cleavage (data not shown). Excess AdoMet was removed and the extent of labelling ascertained by scintillation counting. We ﬁrst analysed two unmodiﬁed DNA substrates; a non-speciﬁc DNA and a speciﬁc DNA carrying the PstII recognition site (Figure 4A). The speciﬁc DNA was modiﬁed to a signiﬁcantly greater degree than the unmodiﬁed DNA (>1.6 · 105 DPM compared to <2.0 · 103 DPM with background at 0.113 · 103, Figure 4B). Secondly we analysed three pre-modiﬁed oligo- nucleotides in which an N6-methyl deoxyadenosine phos- phoramidite was incorporated during synthesis at one of the three target adenine residues in the PstII recogntion sequence (Figure 4A). When the ﬁrst adenine on the 50-CATCAG-30 strand was pre-methylated (position 3), PstII transferred very little radioactivity to the DNA (<7.0 · 103 DPM, Figure 4B). In contrast, pre-methylation of either of the other two adenine residues in the PstII sequence (positions 1 or 2) resulted in labelling at a level similar to that observed on the unmodiﬁed speciﬁc DNA (>1.5 · 105 DPM). Therefore, we conclude that PstII only hemi-methylates its recognition sequence at one