4776 Nucleic Acids Research, 2005, Vol. 33, No. 15 Figure 1. The Res and Mod proteins of PstII. (A) Schematic representation of Mod and Res. The locations of amino acid motifs and putative protein domains are indicated. In Mod the motifs in grey are part of the AdoMet binding region, the motifs in black are part of the catalytic region, and the ‘TRD’ is the Target Recognition Domain. (B) Sequence alignments of Helicase Motif 1 (Walker A) and Region X as indicated for a selection of Type IIIB and IIIA enzymes. Alignments and consensus sequences were obtained using ClustalW (28) using protein sequences from REBASE (1). grey shading represent >80% identity, uppercase characters indicate >60% identity, ‘+’ indicates a hydrophobic residue with >80% identity, ‘o’ indicates a hydrophobic residue with >80% identity, and ‘·’ indicates a variable residue. (C) SDS–PAGE [10% (v/v) polyacrylamide] analysis of protein samples during PstII purification as indicated. Digital images were captured on a Kodak Image Station 440CF without editing and using a linear intensity scale. supplied by MWG Biotech (Germany). Two-site DNA substrates were created by direct cloning of the following duplex oligonucleotide into the appropriate sites of pNAG4 (17) or pAT153 (18): 50-AGCTGACGACCATCAGGGACA- GCTCGAG-30 was annealed to 50- AGCTCTCGAGCTGTC- CCTGATGGTCGTC-30 (PstII sequence underlined), and the duplex cloned into the HindIII site of pNAG4 to give pLJP11a or pLJP11b depending upon the orientation of the PstII site (Figure 1A); 50-AGCTGATGAACTGCCTTGCTACAGTGT- GGCTAGAAC-30 was annealed to 50-AGCTGTTCTAGCCA- CACTGTAGCAAGGCAGTTCATC-30 (PstII sequence underlined), and the duplex cloned into the HindIII site of pAT153 to give pLJP12c (sites chosen to be in indirect repeat); and, 50-AGCTGATGCTTTCTAAGGGTAATTTTAAAAT- ATCT-30 was annealed to 50-AGCTAGATATTTTAAAAT- TACCCTTAGAAAGCATC-30 (PstII sequence underlined), and the duplex cloned into the HindIII site of pAT153 to give pLJP12d (sites chosen to be in indirect repeat). Escherichia coli TOP10 cells (Invitrogen) were transformed with the plasmids and the covalently closed circular (CCC) form of the DNA puriﬁed by density gradient centri- fugation in CsCl-ethidium bromide (19). Where labelled DNA was required, the transformants were grown in M9 minimal medium supplemented with 37 MBq/l [3H- methyl] thymidine. Linear DNA substrates were generated from the plasmid by digestion with the appropriate Type II restriction enzyme and the DNA puriﬁed by phenol/ chloroform and chloroform extraction followed by ethanol precipitation. DNA concentrations were determined from UV absorbance at 260 nm, assuming that an OD1 corres- ponds to 50 mg/ml DNA and a molecular weight of 6.6 · 105 Da/kb.