RESTRICTION BY ECOKI IS ENHANCED BY COOPERATIVE INTERACTIONS BETWEEN TARGET SEQUENCES AND IS DEPENDENT ON DEAD BOX MOTIFS

One subunit of both type I and type III restriction and modification e nzymes contains moths characteristic of DEAD box proteins, which impli es that these enzymes may be DNA helicases. This subunit is essential for restriction, but not modification. The current model for restricti on by both types of enzyme postulates that DNA cutting is stimulated w hen two enzyme complexes bound to neighbouring target sequences meet a s the consequence of ATP-dependent DNA translocation. For type I enzym es, this model is supported by in vitro experiments, but the predicted co-operative interactions between targets have not been detected by a ssays that monitor restriction in vivo. The experiments reported here clearly establish the required synergistic effect but, in contrast to earlier experiments, they use Escherichia coli K-12 strains deficient in the restriction alleviation function associated with the Rac propha ge. In bacteria with elevated levels of EcoKI the cooperative interact ions are obscured, consistent with cooperation between free enzyme and that bound at target sites. We have made changes in three of the moti fs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions. Conservativ e changes in each of the three motifs impair restriction.