A dual role for substrate S-adenosyl-l-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase

AUTOR(ES)

Malygin, Ernst G.

FONTE

Oxford University Press

RESUMO

The fluorescence of 2-aminopurine (2A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target (2A/A duplex) or its methylated derivative (2A/mA duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with 2A being flipped out of the DNA helix. Though neither S-adenosyl-l-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-l-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an 2A/2A double-substituted duplex. Since the 2A/mA duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric 2A/A and 2A/mA duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.

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