BP I, A NEW BCGI-LIKE RESTRICTION-ENDONUCLEASE, WHICH RECOGNIZES A SYMMETRICAL SEQUENCE/

BcgI and BcgI-like restriction endonucleases cleave double stranded DN A specifically on both sides of their asymmetric recognition sequences which are interrupted by several ambiguous base pairs, Their heterosu bunit structure, bifunctionality and stimulation by AdoMet make them d ifferent 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 symmetr ic 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 DN A cleavage and methyltransferase activities, There are two polypeptide s in the homogeneous preparation of BplI with molecular masses of simi lar to 74 and 37 kDa, The sizes of the BplI subunits are close to thos e of BcgI, but the proportion 1:1 in the final preparation is differen t 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 sinefungi n can replace AdoMet in the cleavage reaction, AdoHcy activated BplI y ields complete cleavage of DNA.