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

Abstract

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N6 )-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-Lhomocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme⅐substrate complexes using an Nd 3؉ :yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme⅐substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam⅐AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.

Broad-host-range Salmonella bacteriophage STP4-a and its potential application evaluation in poultry industry

“…Furthermore, gp247 of phage STP4-a showed 75% homology to anti-restriction nuclease of Klebsiella phage KPV15. Then the putative anti-restriction nuclease of phage STP4-a may be resistant to the host R-M system, preventing the host endonuclease from cleaving specific DNA sequences of phages ( Evdokimov et al., 2007 ), and then widen the host range in a sense. Such conclusions were also found in phage PVP-SE1 that a putative DNA cytosine methyltransferase (gp9) may thus methylate cytosine resides at endonuclease sites, which could protect phage DNA from being cut by its hosts' restriction endonucleases in the R-M system ( Santos et al., 2011 ).…”

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit

“…For some related methyltransferases, e.g. Dam enzyme from phage T4 phage, it has been demonstrated that the binding of the methyl group donor or the nucleic acid substrate reciprocally improve the affinity for each other in the ternary complex (42). This suggests that NpmA may also exhibit significantly increased affinity for AdoMet when pre-bound to the 30S subunit.…”

wksl3, a New Biocontrol Agent for Salmonella enterica Serovars Enteritidis and Typhimurium in Foods: Characterization, Application, Sequence Analysis, and Oral Acute Toxicity Study

“…Nuclear disruption of host DNA occurs because of the action of endonucleases, and disrupted nucleotide particles are recombined into progeny phage DNA (76). Methyltransferases or methylases in phages protect DNA from self or host cell restriction enzymes (77). The DNA methylase N-4/N-6 domain protein was encoded in SE2 (42), while other SETP3-like phages did not have homologous genes or putative methylase functional genes in their genomes.…”