Introduction 1,2 3 4,5 in cis 6,7 5,8 9 4,5 10 11,12 3 13 2+ 10,14 S 11,12 14,15 16–18 3 2 9,18 in cis in trans 19–23 24,25 9 24 in trans V max 25 16,18,26 in trans 20,25,27 2 27–29 30 22,31 24,32 33 22,24,34,35 2 3 36 13 36–38 39,40 2 Table 1 41–45 Table 1 3 42,44,46–48 4 42 44 49 50 46–48 2+ Table 1 43 Table 1 3 41,46–50 Table 1 42–45 45 Table 2 Results BcgI 45 Table 1 17,51–53 Supplementary Data, Figure S1 18 Table 3 45 in cis 9,18 33–35,54 54 55 33 34,35 res 21 21 Figure 1 Figure 1 Figure 1 AloI, BaeI and BplI Supplementary Data, Figures S1–S3 Table 3 56 9 40 Table 1 49,50 47 Table 1 Figure 2 Table 3 Figure 2 20,27 in trans 24,25 24,39 Figure 3 Figure 3 in cis in trans in cis in trans 17–19 Other Type IIB enzymes Table 1 Table 2 Table 3 Table 1 Figure 3 Figure 5 Table 3 Figure 4 K m 16,18,26,39 Figure 4 Table 3 Table 2 Figure 2 Figure 4 Figure 4 Figure 4 Table 3 Table 3 Modes of action in cis in cis 20,21 19,22,24 9,25,33 17,57 30,33 16,24 Figure 5 Figure 5 Figure 1 In all three examples, and also with all of the other Type IIB enzymes examined here apart from AloI (data not shown), their reactions yielded none of the OC form of the DNA that would have arisen if products cut in one strand, at one or both sites, had accumulated during the reaction. A SC DNA with two recognition sites for a Type IIB enzyme has eight scissile phosphodiester bonds, in top and bottom strands on either side of both sites. Cleavage of any one of these eight bonds will convert the SC to the OC form but if the next phosphodiester bond to be cleaved is chosen at random from the seven remaining targets, six out of the seven leave the DNA in its OC state and only one, that opposite the initial nick, will result in the OC form being converted to the linear product with one double-strand break. Hence, Type IIB enzymes might be expected to generate large amounts of the OC intermediate before creating any linear product. The observation that no OC DNA accumulated shows instead that, after cutting one strand at a single locus, Type IIB enzymes must cut the second strand at that locus very shortly thereafter, within the lifetime of a single DNA–enzyme complex. Figure 5 Materials and Methods 16,24,33 Figure 5 17,57 Figure 1 Figure 1 in cis 33 in cis, Table 3 Figure 5 Figure 1 Discussion The general case 2 Table 1 Table 3 Figure 4, Table 3 Figure 1 Figure 3 45 in cis in trans 9,24,25 in cis 34,54 in trans 42,49 46–48 viz viz Table 1 9,19–21,33 27–30 22,24,32–35 1,4,5 3 18 36–40 in vivo in vitro 58,59 19 9,38,60 22,35,61,62 61 The special case 19 Figures 2(c), 4(a) and (b) Figure 4 Figure 3 Table 1 42,50 2,3 57 Table 1 50 13,37–40 36 Materials and Methods Enzymes 21 33,34,54 Table 2 6 7 10,19 DNA 63 34 33 18 Supplementary Data, Figures S1–S3 Escherichia coli methyl 3 31–34 21 18,33,54 A 260 Reactions 3 Table 2 24,32,38 26,40 16,25 7,33–35 25,40,57 Figures 1–5 Table 3 17,57 Figure 2 Figure 2 0 0 K m 52 42,45 Supplementary Data Supplementary Figures S1–S3  Supplementary Data doi:10.1016/j.jmb.2006.12.070