R. Sapranauskas et al., Novel subtype of type IIs restriction enzymes - BfiI endonuclease exhibitssimilarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium, J BIOL CHEM, 275(40), 2000, pp. 30878-30885
The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleot
ide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleot
ides downstream of the recognition sequence. The genes coding for the BfiI
restriction-modification (R-M) system were cloned/sequenced and biochemical
characterization of BfiI restriction enzyme was performed. The BfiI R-M sy
stem contained three proteins: two N4-methylcytosine methyltransferases and
a restriction enzyme, Sequencing of bisulfite-treated methylated DNA indic
ated that each methyltransferase modifies cytosines on opposite strands of
the recognition sequence. The N-terminal part of the BfiI restriction enzym
e amino acid sequence revealed intriguing similarities to an EDTA-resistant
nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that
BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of M
g2+ ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphat
e. However, unlike the nonspecific S. typhimurium nuclease, BfiI restrictio
n enzyme cleaves DNA specifically. We propose that the DNA-binding specific
ity of BfiI stems from the C-terminal part of the protein. The catalytic N-
terminal subdomain of BfiI radically differs from that of type II restricti
on enzymes and is presumably similar to the EDTA-resistant nonspecific nucl
ease of S. typhimurium; therefore, BfiI did not require metal ions for cata
lysis. We suggest that BfiI represents a novel subclass of type IIs restric
tion enzymes that differs from the archetypal KokI endonuclease by the fold
of its cleavage domain, the domain location, and reaction mechanism.