Effect of base analog substitutions in the specific GATC site on binding and methylation of oligonucleotide duplexes by the bacteriophage T4 Dam DNA-[N-6-adenine] methyltransferase

The interaction of the phage T4 Dam DNA-[N-6-adenine] methyltransferase wit h 24mer synthetic oligonucleotide duplexes having different purine base sub stitutions in the palindromic recognition sequence, GATC, was investigated by means of gel shift and methyl transfer assays, The substitutions were in troduced in either the upper or lower strand: guanine by 7-deazaguanine (G- ->D) or 2-aminopurine (G-->N) and target adenine by purine (A-->P) or 2-ami nopurine (A-->N). The effects of each base modification on binding/methylat ion were approximately equivalent for both strands. G-->D and G-->N substit utions resulted in a sharp decrease in binary complex formation, This sugge sts that T4 Dam makes hydrogen bonds with either the N7- or O6-keto groups (or both) in forming the complex, In contrast, A-->P and A-->N substitution s were much more tolerant for complex formation. This confirms our earlier observations that the presence of intact 5'-G:C base pairs at both ends of the methylation site is critical, but that base substitutions within the ce ntral A:T base pairs show less inhibition of complex formation. Addition of T4 Dam to a complete substrate mixture resulted in a burst of [H-3]methyla ted product. In all cases the substrate dependencies of bursts and methylat ion rates were proportional to each other. For the perfect 24mer k(cat) = 0 .014/s and K-m = 7.7 nM was obtained, In contrast to binary complex formati on the two guanine substitutions exerted relatively minor effects on cataly tic turnover (the k(cat) was reduced at most 2.5-fold), while the two adeni ne substitutions showed stronger effects (5- to 15-fold reduction in k(cat) ). The effects of base analog substitutions on K-m(DNA) were more variable: A-->P (decreased); A-->N and G-->D (unchanged); G-->N (increased).