Nucleic Acids Research, 2005, Vol. 33, No. 19 6205 Supplementary Data). In vitro translation products were used directly in cleavage reactions. Reaction conditions were as follows: up to 10 ml target DNA and 3 ml in vitro translation reaction were incubated for cleavage as above. For cleavage site mapping one 32P-radiolabelled primer was used in the target PCR and cleavage products were separated on 8% poly- acrylamide gels together with sequencing ladders produced with the fmol DNA Cycle Sequencing System (Promega) using the same labelled primers: nrdB1, nrdB2, D4 and G2 (Supplementary Table 1). Targets used in SegH competition assay were ampliﬁed from T2, using primers Pat11 and G2(F) and from T4 and LZ2 using Pat11 and G4(F) (Supplementary Table 1). Targets used in Hef competition assay were ampliﬁed from T2, T4 and U5 using primers BS10 and A2(F) (Supplementary Table 1). Competition assays with 0.4 and 0.8 mg of partially puriﬁed His-tagged SegH were performed with equimolar amounts of targets (700 fmol of T2, T4 and LZ2) in 20 ml reactions. The cleavage reaction was performed in 66 mM K-acetate, 33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 0.1 mg/ml BSA at 37 C, and samples were withdrawn for analyses as indicated. Competition assays with 5 and 10 ml of in vitro translated Hef were performed in 50 ml reactions with equimolar amounts of targets (700 fmol of T2, T4 and U5). The cleavage reaction Figure 1. (A) Alignment of T4 Seg-proteins and LZ2 SegH. (B) Alignment of the different MobE proteins. The sequence of the MobE protein from Schizo-T-even phage AehI was obtained from the genome sequence, GenBank accession no. AY266303. Residues with black shading and white text are identical in 70% of the sequences, grey shading and black text denotes positions with similar residues in 70% of the sequences. (C) Alignment of a region of Hef (residues 251–345) with a region of a hypothetical protein from Corynebacterium glutamicum (residues 283–370 of 606, GenBank accession no. NP_600985) and a region from a representative ORF with unknown function that appears to be related to a diverse group of endonucleases (residues 9–102 of 144 from Rhodopseudomonas palustris hypothetical protein, GenBank accession no. NP_946397). Residues with black shading are conserved, and asterisks denote highly conserved residues among the diverse group of endonucleases.