Bacteriophage T4Dam DNA-(Adenine-N6)-methyltransferase: COMPARISON OF PRE-STEADY STATE AND SINGLE TURNOVER METHYLATION OF 40-MER DUPLEXES CONTAINING TWO (UN)MODIFIED TARGET SITES

Ernst G Malygin,Bianca Sclavi,Victor V Zinoviev,Alexey A Evdokimov,Stanley Hattman,Malcolm Buckle

doi:10.1074/jbc.m409786200

Abstract

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-l-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex. Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps. With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase. Single turnover methylation of 40-mer duplexes also proceeded in two stages. First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a two-step methylation. Instead of processive movement of T4Dam, a conformational adaptation occurred. We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand. This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. Under single turnover conditions, there was complete methylation of all target adenine residues with each of the two-site 40-mer duplexes.

Highlights

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N6)methyltransferase-mediated methyl group transfer from S-adenosyl-L-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5؅-GATC/5؅GATC) or some modified variant(s)
In contrast to the steady state, here DNAMe1 signifies the departure of T4Dam from a methylated site GMTC1 without physically dissociating from the DNA molecule (M denotes N6-methyladenine, m6Ade). (iv) Following methyl transfer at one site and linear diffusion to a hemimethylated site, T4Dam was capable of rapidly reorienting itself to the unmethylated strand. (v) The inhibition potential of fully methylated sites 5Ј-GMTC/5ЈGMTC was much lower in a long DNA molecule as compared with short single-site duplexes. (vi) The T4Dam structural state depended on the molar ratio of the enzyme/duplex [6]
P is the burst of product normalized to [enzyme], kmeth is the rate of methyl transfer, and kcat is the steady state reaction rate constant

Summary

Introduction

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N6)methyltransferase-mediated methyl group transfer from S-adenosyl-L-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5؅-GATC/5؅GATC) or some modified variant(s). Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand. This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. Since the results summarized above were based primarily on steady state and single turnover methylation studies [5], the present work was undertaken to determine whether methylation under pre-steady state conditions would yield results consistent with the kinetic model we proposed previously [8]

Objectives

Results

Conclusion