TYPE-III RESTRICTION ENDONUCLEASES TRANSLOCATE DNA IN A REACTION DRIVEN BY RECOGNITION SITE-SPECIFIC ATP HYDROLYSIS

Type III restriction/modification systems recognize short non-palindro mic sequences, only one strand of which can be methylated, Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction, We have shown previously that suicidal re striction by the type III enzyme EcoP15I is prevented if all the unmod ified sites are in the same orientation: restriction by EcoP15I requir es a pair of unmethylated, inversely oriented recognition sites, We ha ve now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPas e activity, the potential driving force of DNA translocation, The ATPa se activity is uniquely recognition site-specific, but EcoP15I-modifie d sites also support the reaction, EcoP15I DNA restriction patterns ar e shown to be predetermined by the enzyme-to-site ratio, in that site- saturating enzyme levels elicit cleavage exclusively between the close st pair of head-to-head oriented sites, DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15 I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently track ing EcoP15I-DNA complexes.