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

DNA cleavage by type III restriction endonucleases requires two inversely o riented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterize d the structure and mode of action of the related EcoP1I and EcoP15I enzyme s. Analytical ultracentrifugation and gel quantification revealed a common Res(2)Mod(2) subunit stoichiometry. Single alanine substitutions in the put ative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hy drolysis, whilst a substitution in helicase motif VI abolished both activit ies. Positively supercoiled DNA substrates containing a pair of inversely o riented recognition sites were cleaved inefficiently, whereas the correspon ding relaxed and negatively supercoiled substrates were cleaved efficiently , suggesting that DNA overtwisting impedes the convergence of the transloca ting enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circul ar substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzy me concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP 1I resulted in cutting the bottom DNA strand at the EcoP15I site. These dat a suggest that double-strand breaks result from top strand cleavage by a Re s subunit proximal to the site of cleavage, whilst bottom strand cleavage i s catalysed by a Res subunit supplied in trans by the distal endonuclease i n the collision complex. (C) 2001 Academic Press.