Bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase. Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites

The Journal of Biological Chemistry
E G MalyginMalcolm Buckle

Abstract

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-l-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex. Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps. With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase. Single turnover methylation of 40-mer duplexes also proceeded in two stages. First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a two-step methylation. Instead of processive movement of T4Da...Continue Reading

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Citations

Feb 9, 2006·Nucleic Acids Research·Chad B Thomas, Richard I Gumport
Sep 9, 2006·Microbiology and Molecular Biology Reviews : MMBR·Josep Casadesús, David Low
Nov 3, 2007·Biological Chemistry·Victor V ZinovievStanley Hattman
Feb 27, 2010·Critical Reviews in Biochemistry and Molecular Biology·Urulangodi Kunhiraman Madhusoodanan, Desirazu N Rao
Jan 21, 2012·Critical Reviews in Biochemistry and Molecular Biology·Ernst G Malygin, Stanley Hattman
Oct 22, 2016·Applied Microbiology and Biotechnology·Barbara MaciejewskaZuzanna Drulis-Kawa

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