Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I.

Abstract DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterized the structure and mode of action of the related Eco P1I and Eco P15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res 2 Mod 2 subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. Eco P1I and Eco P15I could co-operate in DNA cleavage on circular substrate containing several Eco P1I sites inversely oriented to a single Eco P15I site; cleavage occurred predominantly at the Eco P15I site. Eco P15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The Eco P1I nuclease mutant greatly stimulated the Eco P15I nicking activity, while the Eco P1I motif VI mutant did not. Moreover, combining an Eco P15I nuclease mutant with wild-type Eco P1I resulted in cutting the bottom DNA strand at the Eco P15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.