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.

Figure 1

Fluorescence analysis of T4 Dam titration of duplexes ²A/A (A) and ²A/^mA (B) in the absence of AdoMet; duplexes were at 100 or 200 nM as indicated. Addition at the last titration point of more duplex (up to 200 nM) to the mixture containing 100 nM of ²A/A resulted in an increase in fluorescence intensity confirming that the fluorescence quenching observed was fully reversible (A). Fluorescence intensity is given in arbitrary units (a.u.), and intrinsic fluorescence of the free duplex was subtracted in this and subsequent figures.

Figure 2

Effect of added AdoMet on fluorescence analysis of T4 Dam titration. Duplexes ²A/A (A), ²A/^mA (B) or ²A/²A (C) were at 200 nM; AdoMet was added to a final concentration of 8 µM at the last titration point. For duplex ²A/A (A) titrations are shown in the absence or presence of the reaction inhibitors AdoHcy (20 µM) or sinefungin (8 µM). The lower curve in (B) corresponds to a titration of the ²A/^mA duplex in the presence of 8 µM AdoMet.

Figure 3

Fluorescence titration of T4 Dam (200 nM) with duplex ²A/A (A), ²A/^mA (B) or ²A/²A (C). At the last titration point in (A) and (B), cofactor AdoMet was added to a final concentration of 8 µM.

Figure 4

Glutaraldehyde crosslinking of T4 Dam (1 µM) in the presence of varying concentrations of duplex: (A) duplex ²A/A; (B) duplex ²A/^mA. Lane 1, T4 Dam, untreated; lane 2, T4 Dam treated with 0.0025% glutaraldehyde; lanes 3–8, T4 Dam treated in the presence of duplex at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 µM, respectively; lane 9, molecular weight standards. (C) Crosslinking in the presence of 5 µM AdoMet. Lane 1, T4 Dam, untreated; lane 2, T4 Dam + AdoMet, treated with 0.0025% glutaraldehyde; lanes 3–8, T4 Dam + AdoMet, treated in the presence of the ²A/^mA duplex at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0 and 4.0 µM, respectively; lane 9, molecular weight standards.

Figure 5

Time course of T4 Dam methylation of duplex ²A/A. T4 Dam and duplex ²A/A concentrations were 0.158 and 1 µM (A) or 2.7 and 0.2 µM (B), respectively. T4 Dam was pre-incubated with 8 µM of [³H]AdoMet in both cases. In (A) the number of CH₃ groups transferred per molecule of enzyme bound was plotted (multiple turnover conditions where DNA was in excess); in (B) the number of CH₃ groups transferred per bound duplex was plotted (single turnover conditions where enzyme was in excess).