Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium.
The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides 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 system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme 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 Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity 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 restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.
Sapranauskas
R
R
Institute of Biotechnology, Graiciuno 8, Vilnius 2028, Lithuania.
Sasnauskas
G
G
Lagunavicius
A
A
Vilkaitis
G
G
Lubys
A
A
Siksnys
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000145:classification*; D018076:DNA, Complementary / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004720:Endonucleases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008836:Micrococcal Nuclease; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013447:Sulfites / Q000494:pharmacology; D013997:Time Factors
D001426:Bacterial Proteins; D018076:DNA, Complementary; D009596:Nitrophenols; D009841:Oligonucleotides; D013447:Sulfites; C074002:nuc protein, staphylococcus; C002887:bis(4-nitrophenyl)phosphate; D003596:Cytosine; D004492:Edetic Acid; D015257:DNA-Cytosine Methylases; D004720:Endonucleases; D004262:DNA Restriction Enzymes; C113635:endodeoxyribonuclease BfiI; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; D008836:Micrococcal Nuclease; D008274:Magnesium; C009279:sodium bisulfite