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Restriction and modification of deoxyarchaeosine (dG+)-containing phage 9 g DNA
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Restriction and modification of deoxyarchaeosine (dG+)-containing phage 9 g DNA
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Restriction and modification of deoxyarchaeosine (dG+)-containing phage 9 g DNA
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Journal Article
Restriction and modification of deoxyarchaeosine (dG+)-containing phage 9 g DNA
2017
Overview
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
5m
C or
N
6m
A 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.
