Identification and biosynthesis of thymidine hypermodifications in the genomic DNA of widespread bacterial viruses.

Abstract

Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the <i>Delftia</i> phage ΦW-14 and the <i>Bacillus</i> phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the <i>Pseudomonas</i> phage M6 and the <i>Salmonella</i> phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) <i>Nucleic Acids Res</i> 41:7635-7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-<i>N</i>e<i>O</i>mdU) and 5-(2-aminoethyl)uridine (5-<i>N</i>edU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-<i>N</i>e<i>O</i>mdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.