Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)10/8downarrow

Abstract

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end. Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage. S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3'. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

S-Adenosyl-L-methionine–Dependent Restriction Enzymes

“…ATP does not affect the endonucleolytic activity of the enzyme. Analogs of AdoMet such as S-adenosyl homocysteine (AdoHcy) and sinefungin (Figure 4) could replace AdoMet as cleavage cofactor, indicating that binding of cofactor is essential for cleavage (Jurenaite-Urbanaviciene et al, 2001). MmeI cleaves DNA 20/18 bases away from the sequence, 5 -TCCRAC (where R strands for purine).…”

Chemically-Induced Affinity Star Restriction Specificity: a Novel TspGWI/sinefungin Endonuclease With Theoretical 3-bp Cleavage Frequency

“…Among them, a growing group of atypical type II REases, represented by bifunctional enzymes with REase and MTase activities within a single polypeptide (subtype IIC) is worth mentioning. Examples of this group include Eco 57I (7,8), Hae IV (9), Alo I (10), Bse MII (11), enzymes from the Mme I family (12,13), and enzymes from the Thermus sp. family (1417).…”

Wheat Endonuclease WEN1 Dependent on S-Adenosyl-L-Methionine and Sensitive to DNA Methylation Status

“…Thus, S-adenosyl group of S-AdoMet, S-AdoHcys and SiBA is an effector that modulates WEN1 activity. This is the first case to show that plant endonuclease activity like that of some typical bacterial restriction endonucleases 17,18 is modulated by S-AdoMet and its analogs.…”