Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs.

Abstract

One subunit of both type I and type III restriction and modification enzymes contains motifs characteristic of DEAD box proteins, which implies that these enzymes may be DNA helicases. This subunit is essential for restriction, but not modification. The current model for restriction by both types of enzyme postulates that DNA cutting is stimulated when two enzyme complexes bound to neighbouring target sequences meet as the consequence of ATP-dependent DNA translocation. For type I enzymes, this model is supported by in vitro experiments, but the predicted co-operative interactions between targets have not been detected by assays 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 prophage. In bacteria with elevated levels of EcoKI the co-operative interactions are obscured, consistent with co-operation between free enzyme and that bound at target sites. We have made changes in three of the motifs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions. Conservative changes in each of the three motifs impair restriction.