Phage T4 DNA [N ⁶ -Adenine] Methyltransferase: Kinetic Studies Using Oligonucleotides Containing Native or Modified Recognition Sites

Abstract

The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.

Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

“…After the chemical step of methyl group transfer from AdoMet to DNA, the methylated duplex dissociates relatively rapidly (k 3 ϭ 1.7 s Ϫ1 ). The dissociation of the second product, AdoHcy, appears to be the rate-limiting step of the reaction and proceeds with k 4 ϭ 0.018 s Ϫ1 ; this agrees with the value of k cat ϭ 0.015 s Ϫ1 determined previously (37). The release of AdoHcy from the FH complex can occur by two pathways.…”

“…AdoMet and 20-mer duplex DNA. The substrate concentrations used in these experiments were up to 5-fold above K m for AdoMet (K m AdoMet ϭ 490 nM) and 15-fold above K m for DNA (K m DNA ϭ 6.3 nM) (37). As shown in Fig.…”

“…T4 Dam reactions were carried out at 25°C in buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 0.2 mg/ml bovine serum albumin (36). A low concentration of T4 Dam (1 nM) was used for most experiments to increase the accuracy of the calculation of the initial reaction velocities by reducing the influence of any burst in product formation (37). The concentrations of AdoMet, substrate DNA, and inhibitors varied according to the individual experiment.…”

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

“…1 and Table III). The k cat values for 40-mer duplexes A-D (with two potential sites for methylation) and the corresponding mixtures of constituents were all comparable; they were close to the value of k cat ϭ 0.015 s Ϫ1 observed for a single-site 20-mer duplex (20). Hence, it appears that T4 Dam steady state methylation of 40-mer duplexes is similar to the methylation of a shorter 20-mer duplex, and both have the same rate-limiting step in the overall reaction.…”

“…DNA MTase Assay-DNA MTase assays were similar to those reported previously (20). T4 Dam reactions were carried out at 25°C in buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 0.2 mg/ml bovine serum albumin (17).…”