ECOKI WITH AN AMINO-ACID SUBSTITUTION IN ANY ONE OF 7 DEAD-BOX MOTIFSHAS IMPAIRED ATPASE AND ENDONUCLEASE ACTIVITIES

For type I restriction systems, recently determined nucleotide sequenc es predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR), The conserved sequences empha size motifs characteristic of the DEAD-box family of proteins which co mprises putative helicases, and they identify a new candidate for moti f IV. We provide evidence based on an analysis of EcoKI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV. Amino acid substitutions within t he newly identified motif IV and those in six other previously identif ied DEAD-box motifs, but not in the original motif IV, confer restrict ion-deficient phenotypes. We have examined the relevance of the DEAD-b ox motifs to the restriction pathway by determining the steps permitte d in vitro by the defective enzymes resulting from amino acid substitu tions in each of the seven motifs, EcoKI purified from the seven restr iction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conforma tional change essential for the pathway of events leading to DNA cleav age, The seven enzymes have little or no ATPase activity and no endonu clease activity, but they retain the ability to nick unmodified DNA, t hough at reduced rates. Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP- dependent translocation of DNA, particularly if this involves DNA heli case activity.