BplI, a new BcgI-like restriction endonuclease, which recognizes a symmetric sequence.
Bcg I and Bcg I-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 Bcg I-like restriction endonuclease, Bpl I from Bacillus pumilus , which in contrast to all other Bcg I-like enzymes, recognizes a symmetric interrupted sequence, and which, like Bcg I, cleaves double stranded DNA upstream and downstream of its recognition sequence (8/13)GAGN5CTC(13/8). Like Bcg I, Bpl I is a bifunctional enzyme revealing both DNA cleavage and methyltransferase activities. There are two polypeptides in the homogeneous preparation of Bpl I with molecular masses of approximately 74 and 37 kDa. The sizes of the Bpl I subunits are close to those of Bcg I, but the proportion 1:1 in the final preparation is different from that of 2:1 in Bcg I. Low activity observed with Mg2+increases >100-fold in the presence of AdoMet. Even with AdoMet though, specific cleavage is incomplete. S -adenosylhomocysteine (AdoHcy) or sinefungin can replace AdoMet in the cleavage reaction. AdoHcy activated Bpl I yields complete cleavage of DNA.
Vitkute
J
J
Institute of Biotechnology, Graiciuno 8, 2028 Vilnius, Lithuania.
Maneliene
Z
Z
Petrusyte
M
M
Janulaitis
A
A
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D001407:Bacillus / Q000201:enzymology*; D010582:Bacteriophage lambda / Q000235:genetics; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000494:pharmacology; D013379:Substrate Specificity
D004279:DNA, Viral; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; C073736:endodeoxyribonuclease BcgI; C109633:endodeoxyribonuclease BplI; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D000241:Adenosine; C006235:sinefungin