Bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase): evidence for two distinct stages of methylation under single turnover conditions.

We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA << 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (kmeth1 = 0.21 s-1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (kmeth2 = 0.023 s-1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (kmeth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (kmeth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.