Methylation by a mutant T2 DNA [N-6-adenine] methyltransferase expands theusage of RecA-assisted endonuclease (RARE) cleavage

摘要

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 Bell and EcoRV endonucleases has been shown on phage lambda DNA and with Soil and DpnII endonucleases on yeast chromosomal DNA embedded in agarose.