Introduction 1 2 2+ 3 4 , 5 6–8 8–14 in vitro 15 16–20 21 22 23 17 , 22 24 , 25 26–30 26 , 27 31 26 , 27 32 33–35 36 , 37 36 36,38 39 28 24 40–42 29 43 , 44 19 20 cis trans cis trans 39 , 45 26 , 28 , 29 , 34 44 24 , 25 , 28 , 40 Figure 1 46 47 46 25 41 , 42 41 42 Results Subunit communications in SfiI 25 Figure 1 2 2 46 2 40 25 2 2 25 Figure 1 48 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Enzyme stability and quaternary structure Materials and Methods 2 26 , 32 2 2 2 2+ Table 1 49 M r M r M r Figure 2 M r DNA-binding studies Figure 3 36 Table 1 36,37 2+ 2+ 10–12 Figure 3 Figure 3 The intermediate complex, with a mobility in between that with HEX-21 alone and that with HEX-35 alone, must contain one molecule of HEX-21 and one molecule of HEX-35. The complexes with the highest and the lowest mobilities therefore correspond, respectively, to the SfiI tetramer bound to two molecules of HEX-21 and to two molecules of HEX-35. Both Y68F and wt SfiI can thus form complexes that contain two DNA duplexes bound to a tetrameric protein. However, both proteins were saturated at the DNA concentrations used here, so it remains to be determined whether there are any differences in affinity and/or cooperativity between wt and mutant proteins. Cleavage of two-site plasmid 27 cis 33–35 32 26 E 0 S Figure 4 Figure 4 Figure 4 V max 26 , 27 Figure 4 K m Figure 3 32 50 32 Figure 4 32 cis 33,35 Cleavage of one-site plasmid 26 , 32 , 33 26 Figure 5 Figure 5 Figure 4 − 1 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 39 cis trans 36 , 38 in trans 20 , 44 trans 17,26,28,30 trans 26 Figure 6 Table 1 Figure 5 Figure 6 Figure 6 Figure 5 trans trans 41 , 42 Reactions at individual sites 41 , 42 Table 1 32 trans Figures 3(a) and 6(a) Figure 7 − 1 − 1 32 36 − 1 Figure 7 Figure 7 Figure 5 Discussion 26 , 34 39 24 , 28 25 31 33 36 , 38 41,42 Figure 1 25 cis Figures 2, 3 and 4 Figure 4 Figure 5 trans cis Figures 6 and 7 Figure 6 versus Kinetic model for SfiI cooperativity 51 , 52 Figure 8 S 2+ 2+ 36 in trans S S S S in cis S S S S in cis 39 , 45 S Figure 8 S S S S S S S S S Figure 8 k 1 k − 1 k 2 k − 2 k 3 k 4 k 2 cis trans trans cis 45 Figure 8 Figure 8 Figures 4 and 5 S k 1 k − 2 S k − 1 k 2 k 2 S k 3 Figure 5 k 2 k 3 k 4 k 4 S S Figure 4 k 3 S S Figure 8 S S S S S S S S in trans k 3 S S S S k 2 S S S S S S S G S S trans Structural model for SfiI cooperativity S Figure 8 S S 25 S S Figure 8 Figure 1 1 , 4 , 5 S S Figure 8 25 53 S S S Figure 8 S S S S S Materials and Methods Mutagenesis + + Escherichia coli + + E. coli + + Proteins and DNA E. coli + + 26 − 1 − 1 34 M r 48 26 E. coli methyl 3 32–35 Table 1 Table 1 32 30 , 36 Analytical ultracentrifugation 44 2 r A 539 49 A r = A 0 exp [ M ( 1 − ν ¯ ρ ) ( r 2 − r 0 2 ) ( ω 2 / 2 R T ) ] + B M M r A r A 0 r r 0 B 44 DNA-binding studies 2 2 36 Enzyme assays 3 2 2 2 2 2 18–20 32–36 Figure 5 Figure 8 3 2 Immobilised oligonucleotides 32 41 2 2 32