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Nucleic Acids Research, 2001, Vol. 29, No. 7 1484-1490
© 2001 Oxford University Press

Methylation by a mutant T2 DNA [N6-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage

Irina Minko, Stanley Hattman1, R. Stephen Lloyd and Valeri Kossykh*

Sealy Center for Molecular Science, University of Texas Medical Branch, 5.144 Medical Research Building, Galveston, TX 77555-1071, USA and 1Department of Biology, University of Rochester, Rochester, NY 14627-0211, USA

Properties of a mutant bacteriophage T2 DNA [N6-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 kcat 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 BclI and EcoRV endonucleases has been shown on phage {lambda} DNA and with BclI and DpnII endonucleases on yeast chromosomal DNA embedded in agarose.

* To whom correspondence should be addressed. Tel: +1 409 772 2181; Fax: +1 409 772 1790; Email: valkoss{at}scms.utmb.edu


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