Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/.
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.
Jurenaite-Urbanaviciene
S
S
Institute of Biotechnology and MBI Fermentas, Graiciuno 8, 2028 Vilnius, Lithuania.
Kazlauskiene
R
R
Urbelyte
V
V
Maneliene
Z
Z
Petrusyte
M
M
Lubys
A
A
Janulaitis
A
A
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021122:Protein Subunits; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D021122:Protein Subunits; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; C427309:modification methylase BseMII; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C427307:BseMII endonuclease; D004262:DNA Restriction Enzymes; C054219:endodeoxyribonuclease Eco57I; D008274:Magnesium; D000241:Adenosine; C006235:sinefungin