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

Abstract

The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A 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 (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A 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 (2)A/A and (2)A/(m)A 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.

Bacteriophage T4 Dam DNA-(N 6-adenine)-methyltransferase

“…In this regard, formation of T4 Dam dimers cannot explain these results, because the calculated burst values would have to be reduced 2-fold to account for the binding of two enzyme molecules. Moreover, under the conditions similar to those used in these experiments, we found no evidence for the formation of appreciable amounts of T4 Dam dimers (13).…”

“…Reorientation of T4 Dam to the Methylation Target-We proposed earlier that AdoMet induces an allosteric T4 Dam conformational change, allowing a rapid reorientation of the enzyme to the DNA strand that contains the unmethylated GATC (8,13). Thus, when comparing bursts for different duplexes, it is important to keep in mind that, according to our model, only T4 Dam-AdoMet is capable of reorientation at a hemimethylated site.…”

“…These studies permitted us to characterize the interaction of T4 Dam with one specific site as well as the influence of defined modifications in site structure on kinetic parameters. In addition, we used fluorescence titration to investigate the interaction of T4 Dam with these 20-mer oligonucleotide duplexes in which one or two target Ade residues were substituted by 2-aminopurine (N), and we showed that AdoMet plays a crucial role in T4 Dam reorientation about the DNA duplex (13). The intensity of 2-aminopurine (N) residue fluorescence is low inside the double-stranded DNA helix; however, it increases sharply if the fluorophor is out of stacking interaction, permitting one to study the base flipping process induced by enzyme binding (14).…”

“…The intensity of 2-aminopurine (N) residue fluorescence is low inside the double-stranded DNA helix; however, it increases sharply if the fluorophor is out of stacking interaction, permitting one to study the base flipping process induced by enzyme binding (14). The addition of T4 Dam at a saturating concentration to an unmethylated target (N/A duplex) or its methylated derivative (N/M duplex) resulted in an up to 50-fold increase in fluorescence (13). This indicated that T4 Dam binding promotes or stabilizes base (nucleoside) flipping out of the DNA helix.…”

Structure of the bacteriophage T4 DNA adenine methyltransferase

“…Indirect evidence for flipping of the target adenine by T4Dam has been obtained by fluorescence analysis with substrates containing the base analog 2-aminopurine 37 . Encouraged by the similar conformations of the Asp-Pro-Pro-Tyr motif in all N6-adenine MTase structures, we superimposed the target adenine of M.TaqI onto T4Dam (Fig.…”