Bp/I, a new BcgI-like restriction endonuclease, which recognizes a symmetric sequence

BcgI and BcgI-like restriction endonucleases cleave double stranded DNA specifically on both sides of their asymmetric recognition sequences which are interrupted by several ambiguous base pairs, Their heterosubunit structure, bifunctionality and stimulation by AdoMet make them different from other classified restriction enzymes, Here we report on a new BcgI-like restriction endonuclease, BplI from Bacillus pumilus, which in contrast to all other BcgI-like enzymes, recognizes a symmetric interrupted sequence, and which, like BcgI, cleaves double stranded DNA upstream and downstream of its recognition sequence (8/13) GAGN(5)CTC(13/8), Like BcgI, BplI is a bifunctional enzyme revealing both DNA cleavage and methyltransferase activities, There are two polypeptides in the homogeneous preparation of BplI with molecular masses of similar to 74 and 37 kDa, The sizes of the BplI subunits are close to those of BcgI, but the proportion 1:1 in the final preparation is different from that of 2:1 in BcgI, Low activity observed with Mg2+ increases >100-fold in the presence of AdoMet. Even with AdoMet though, specific cleavage is incomplete, S-adenosyl-homocysteine (AdoHcy) or sinefungin can replace AdoMet in the cleavage reaction, AdoHcy activated BplI yields complete cleavage of DNA.