nitrophenols and sodium-bisulfite

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.

Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Cloning, Molecular; Cytosine; Deoxyribonucleases, Type II Site-Specific; DNA Methylation; DNA Restriction Enzymes; DNA-Cytosine Methylases; DNA, Complementary; Dose-Response Relationship, Drug; Edetic Acid; Endonucleases; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Magnesium; Micrococcal Nuclease; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrophenols; Oligonucleotides; Plasmids; Protein Folding; Protein Structure, Tertiary; Salmonella typhimurium; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sulfites; Time Factors