The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli

Abstract

The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.

Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

“…Allosteric activation by methylated DNA was observed with human Dnmt1 MTase (9,20). The binding of two AdoMet molecules was reported for the EcoDam (21) and PvuII MTases (22,23), and an effector role was suggested for the second AdoMet molecule. The precise binding stoichiometry of AdoMet, as well as its double role as methyl donor and allosteric effector, remains to be fully characterized for T4 Dam (24,25).…”

Dam methyltransferase from Escherichia coLi: sequence of a peptide segment involved in S-adenosyl-methionine binding

“…AdoMet probably binds to Dam methylase at two different sites, as has already been published (21,22). It is assumed to act as an allosteric effector when bound to the allosteric site and as a methyl donor when bound to the catalytic site.…”

“…It is assumed to act as an allosteric effector when bound to the allosteric site and as a methyl donor when bound to the catalytic site. The reaction conditions used for the crosslinking experiments described in this article exclude the possibility of AdoMet binding to the allosteric site, because at salt concentrations exceeding 200 mM NaCl the allosteric effect of AdoMet is lost (21). In the crosslinking experiment, radioactively labelled AdoMet could be replaced in the complex by the competitive inhibitors AdoHcy and sinefungine (17).…”

DNA substrate specificity of pea DNA methylase

“…This complex could be broken either by the reversal of the formation reaction (probably an unlikely event) or on transfer of a methyl group, with the concomitant release of S-adenosylhomocysteine ( Figure 8); that is, the covalent complex will form only transiently in the presence of a methyl donor, but may be stabilized in the presence of analogues such as S-adenosylhomocysteine or synefungin, which do not allow the transfer of a methyl group to occur. Such a situation has been shown to occur with several bacterial methyltransferases (for example, Bergerat and Guschlbauer, 1990;Dubey and Roberts, 1992).…”