4778 Nucleic Acids Research, 2005, Vol. 33, No. 15 Tris-acetate (pH 7.9), 10 mM Mg acetate, 50 mM K acetate, 1 mM DTT] supplemented with AdoMet and NTPs as indic- ated in the text. When PstII was added the buffer was further supplemented with 0.02% (v/v) Triton-X100. Reactions were started by addition of Type III enzyme(s) to reduce the effect of co-methylation upon pre-incubation. Reaction aliquots were quenched after the required incubation times by addition of 0.5 vol of STEB [0.1 M Tris–HCl (pH 7.5), 0.2 M EDTA, 40% (w/v) sucrose, 0.4 mg/ml bromophenol blue]. The CCC DNA substrate, open circle/nicked intermediate (OC) and full- length linear product (FLL) were separated by agarose gel electrophoresis and the percentage of DNA in each band evaluated by scintillation counting (19). Where shown, digital images of the agarose gels were captured on a Kodak Image Station 440CF without editing and using a linear intensity scale. DNA methylation with PstII To generate substrates for methylation studies, oligonuc- leotides were obtained from MWG Biotech. Two of these were non-methylated whilst three were methylated at one of the three putative target adenine residues in the PstII recognition sequence. 50-AGATGACGACCATCAGGGACAGCTTCA- AGGATCGCTCGCGGCTCTTACC-30 was annealed to 50- GGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCT- GATGGTCGTCATCT-30 (PstII sites underlined, methylated adenine resides bold type). Methylation reactions contained 50 mM duplex oligonucleotide and 129 nM PstII mixture in NEB 4 supplemented with 0.465 mM [3H-methyl] AdoMet and 0.02% (v/v) Triton-X100. Reactions were incubated for 3 h at 37 C. The DNA was phenol chloroform extracted and ethanol precipitated and the extent of [3H-methyl] transfer quantiﬁed by scintillation counting. Identification of the PstII recognition sequence An arbitrary plasmid library was obtained as target DNA sub- strates (unpublished data) and digested with PstII for 1 h at 37 C with 4 mM ATP. For those plasmids which were cleaved, the locations of the break sites were mapped to within ±100 bp following secondary cleavage with suitably located Type II restriction enzymes. Computer analysis using ‘SequenceMap’ (Allan Brewer, University of Bristol) was then undertaken to compare all 5 or 6 bp DNA sequences common to the cleavage regions. Of the candidates identiﬁed we rejected all the directly-repeated sites and all indirectly-repeated sites that appeared on substrates which could not be cut. This narrowed down the search to three 6 bp sequences. A second plasmid library was then methylated by PstII in the presence of [3H- methyl] AdoMet and in the absence of ATP. The circular DNA were cleaved with suitably located Type II restriction enzymes to generate a characteristic ladder of linear DNA and these scintillation fragments counted following separation by gel electrophoresis. The various patterns of labelled fragments obtained could only be explained by methylation of the CTGATG sequence. Final conﬁrmation came from analysis of speciﬁc plasmid and oligonucleotide substrates (Figure 2). Figure 2. DNA sequences and substrates for the PstII restriction enzyme. (A) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. (B) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3). (C) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.