Restriction and modification of deoxyarchaeosine (dG⁺)-containing phage 9?g DNA.

ABSTRACT: E. coli phage 9?g contains the modified base deoxyarchaeosine (dG⁺) in its genome. The phage encodes its own primase, DNA ligase, DNA polymerase, and enzymes necessary to synthesize and incorporate dG⁺. Here we report phage 9?g DNA sensitivity to >200 Type II restriction endonucleases (REases). Among the REases tested approximately 29% generated complete or partial digestions, while the remaining 71% displayed resistance to restriction. Phage 9?g restriction fragments can be degraded by DNA exonucleases or ligated by T3 and T4 DNA ligases. In addition, we examined a number of cytosine and adenine methyltransferases to generate double base modifications. M.AluI, M.CviPI, M.HhaI, and M.EcoGII were able to introduce ^5mC or ^N6mA into 9?g DNA as confirmed by partial resistance to restriction and by liquid chromatography-mass spectrometry. A number of wild-type E. coli bacteria restricted phage 9?g, indicating natural restriction barriers exist in some strains. A BlastP search of GenBank sequences revealed five glutamine amidotransferase-QueC homologs in Enterobacteria and Pseudomonas phage, and distant homologs in other phage and bacterial genomes, suggesting that dG⁺ is not a rare modification. We also mapped phage 9?g DNA packaging (pac) site containing two 21-bp direct repeats and a major terminase cleavage site in the phage genome.

SUBMITTER: Tsai R 

PROVIDER: S-EPMC5567051 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature