Bacteriophage DNA thymidine hypermodification biosynthesis is identified via an amino acid‐modified nucleobase intermediate

Abstract

Double stranded DNA viruses of bacteria (i.e. bacteriophages) contain the greatest diversity of modified nucleotides in their DNA. These non‐canonical nucleotides are appear to completely replace one of the canonical bases or substitute for a subset of one of them. Base modifications expand the properties of DNA, conferring additional essential functions such as DNA protection and epigenetic gene regulation.Recently, we have discovered and chemically characterized two new naturally occurring thymidine modifications: 5‐aminoethyl‐2′‐deoxyuridine from Pseudomonas phage M6 and 5‐(2‐aminoethoxy)thymidine from Salmonella phage ViI. These phage DNA thymidine modifications begin from 5‐hydroxymethyl‐2′‐deoxyuridine (5hmdU), where the C‐5 of the heterocycle is covalently modified with substituents. We have identified the subsequent steps in the biosynthesis of these novel hypermodified bases and in turn have been able to partially reconstitute their formation in vitro. Briefly, bioinformatic comparisons of hypemodifying and canonical phage genomes were used to determine candidate hypermodifiying function‐encoding genes. We developed an assay utilizing bead immobilized DNA substrates and in combinations of lysates containing recombinantly expressed candidate hypermodifying genes which allowed us to examine base modification activities in a systematic and reproducible fashion. By this approach, we have discovered that bacteriophage M6 and ViI thymidine DNA modification occurs through three steps. First, a bacteriophage encoded DNA kinase transfers the γ‐phosphate from adenosine triphosphate (ATP) to the 5hmdU on the DNA forming 5PmdU and activating the adjacent hydroxymethyl group at the C5. Second, a novel enzymatic activity ‐‐ amino acid transferase ‐‐ displaces the phosphate with the amino acid; glycine in the case of M6 and serine in the case of ViI. Lastly, a final processing step catalyzed by a pyridoxal phosphate dependent enzymes (PLPDE) or radical s‐adenosylmethionine (rSAM) enzymes leads to the decarboxylation of the amino acid decoration, resulting in the final bacteriophage DNA thymidine modifications. We are currently investigating the individual enzyme mechanisms (particularly on the novel amino acid transferase) to understand the thorough biochemical basis of the phage DNA thymidine hypermodification processes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.