The DNA translocation and ATPase activities of restriction-deficient mutants of EcoKI

EcoKI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase , endonuclease and DNA translocation activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction . These activities involve Am-dependent DNA translocation and DNA cleavage. Mutations that change amino acids within recognisable motifs in HsdR impai r restriction. We have used an in vivo assay to monitor the effect of these mutations on DNA translocation. The assay follows the EcoKI-dependent entr y of phage T7 DNA from the phage particle into the host cell. Earlier exper iments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases s everely impair the ATPase activity of purified enzymes. We find that the mu tations abolish DNA translocation in vivo. This provides evidence that thes e motifs are relevant to the coupling of ATP hydrolysis to DNA translocatio n. Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been sh own to abolish DNA nicking activity. When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability t o hydrolyse ATP and to translocate DNA at wild-type levels. It has been spe culated that nicking may be necessary to resolve the topological problems a ssociated with DNA translocation by type I restriction and modification sys tems. Our experiments show that loss of the nicking activity associated wit h the endonuclease motif of EcoKI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo. (C) 1999 Academic Press.