DAM METHYLTRANSFERASE FROM ESCHERICHIA-COLI - KINETIC-STUDIES USING MODIFIED DNA OLIGOMERS - NONMETHYLATED SUBSTRATES

Steady-state kinetics of the N6-adenine Dam methyltransferase have bee n measured using as substrates non-self-complementary tetradecanucleot ide duplexes that contain the GATC target sequence. Modifications in t he GATC target sequence of one or both of the strands included substit ution of guanine by hypoxanthine, thymine by uracil or 5-ethyl-uracil and adenine by diamino-purine (2-amino-adenine). Thermodynamic paramet ers for the 14-mer duplexes were also determined. DNA methylation of d uplexes containing single dI for dG substitution of the Dam recognitio n site was little perturbed compared with the canonical substrate. Rep lacement of dG residues by dl in both strands resulted in a decrease o f the specificity constant. Substitution in both strands appears to be cumulative. Substitution of the methyl-accepting adenine residues by 2-amino-adenine resulted in surprisingly little perturbation. Dam meth yltransferase is rather tolerant to different substitutions. The resul ts show much less spread than those for the analogous hemimethylated s ubstrates studied previously (Marzabal et al., 1995). The absence of t he methylation marker appears to be deleterious to the specificity of the transition state of the active complex, while the binding of the D NA substrate to the enzyme appears to be mostly determined by the ther modynamic stability of the DNA duplex.