Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage.
Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.
Minko
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Sealy Center for Molecular Science, University of Texas Medical Branch, 5.144 Medical Research Building, Galveston, TX 77555-1071, USA.
Hattman
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Lloyd
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Kossykh
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D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins
D004271:DNA, Fungal; D004279:DNA, Viral; D009841:Oligonucleotides; D014764:Viral Proteins; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499296:dam protein, Bacteriophage T2; C499297:dam protein, Bacteriophage T4; D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes