Bacteriophage T4Dam DNA-(Adenine-N6)-methyltransferase

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 twostep 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.DNA methylation is extremely important for the control of such processes as transcription, genomic imprinting, regulation of development, restoration of the correct structure of DNA, and chromatin organization (1). In contrast to most prokaryotic DNA methyltransferases (MTases), 1 the Dam MTase is not a component of a restriction-modification system; rather, Dam methylation has other important cellular functions such as methyl-directed mismatch repair (reviewed in Ref.2) and bacterial virulence (3). In the latter instance, despite the presence of a large number of virulence factors for intestinal bacteria (Ͼ20), it was sufficient to switch off the gene for the Dam DNA MTase to obtain avirulent live vaccine. Like all natural MTases, Dam MTases use the universal donor, Sadenosyl-L-methionine (AdoMet), transferring methyl groups to the Ade residues in the palindromic sequence 5Ј-GATC-3Ј (1).Earlier, we carried out kinetic analyses of T4Dam-mediated methyl group transfer to oligodeoxynucleotide duplexes containing one or two specific GATC sites with different combinations of (un)methylated targets (reviewed in Ref.

Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes

“…Restriction–modification systems protect the host from foreign invading most often unmodified DNA, such as promiscuous plasmids or infecting bacteriophage. The most common post-replicative base methylations are N6-methyladenine by DNA adenine-N6-methyltransferases (MTases) and 5-methylcytosine modified by cytosine-C5-MTases, which are found in both prokaryotes and eukaryotes, as well in phages as the mechanism of resistance to bacterial RM systems (Malygin et al 2004; Labrie et al 2010). Methylation of infectious phage may also confer protection against other host restriction systems or modify the expression of viral genes for expression in other hosts (Smith and Jeddeloh 2005).…”

Study of Bacteriophage T4-encoded Dam DNA (Adenine-N6)-methyltransferase Binding with Substrates by Rapid Laser UV Cross-linking

“…Another important observation is that methylation of DNAs having more than one specific site is affected by the enzyme's processivity (14), i.e. its ability to undergo facilitated linear diffusion along the DNA and to carry out multiple turnovers on the same substrate molecule.…”