Pre-steady state kinetics of bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase: interaction with native (GATC) or modified sites
AUTOR(ES)
Malygin, Ernst G.
FONTE
Oxford University Press
RESUMO
The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenine [generating N6-methyladenine and S-adenosyl-l-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant kmeth for unmethylated and hemimethylated substrates (0.56 and 0.47 s–1, respectively) was at least 20-fold larger than the overall reaction rate constant kcat (0.023 s–1). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit kmeth. Preformed T4 Dam MTase–DNA complexes are less efficient than preformed T4 Dam MTase–AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=113137Documentos Relacionados
- Interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with oligonucleotides containing native or modified (defective) recognition sites.
- The bacteriophage T2 and T4 DNA-[N6-adenine] methyltransferase (Dam) sequence specificities are not identical.
- Effect of base analog substitutions in the specific GATC site on binding and methylation of oligonucleotide duplexes by the bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase.
- Single amino acid changes that alter the DNA sequence specificity of the DNA-[N6-adenine] methyltransferase (Dam) of bacteriophage T4.
- A dual role for substrate S-adenosyl-l-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase