You are here

Bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase. Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites.

TitleBacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase. Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites.
Publication TypeJournal Article
Year of Publication2004
AuthorsMalygin, EG, Sclavi, B, Zinoviev, VV, Evdokimov, AA, Hattman, S, Buckle, M
JournalJ Biol Chem
Volume279
Issue48
Pagination50012-8
Date Published2004 Nov 26
ISSN0021-9258
KeywordsAdenine, Bacteriophage T4, DNA, DNA Methylation, Kinetics, S-Adenosylmethionine, Site-Specific DNA-Methyltransferase (Adenine-Specific), Time Factors, Tritium
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.

DOI10.1074/jbc.M409786200
Alternate JournalJ. Biol. Chem.
PubMed ID15375160
Grant ListGM29227 / GM / NIGMS NIH HHS / United States
R03 TW05755 / TW / FIC NIH HHS / United States