Type II Restriction of 2-Aminoadenosine (dZ)-Modified DNA and Production of dZ-Modified Plasmid in E. coli

The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host-encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Here, we report the activity results of 147 REases on dZ-modified PCR DNA. Among the enzymes tested, 53% are resistant or partially resistant, and 47% are sensitive when their restriction sites contain one to six modified bases. Sites with four to six dZ substitutions are most likely to resist Type II restriction. Our results support the notion that dZ-modified phage genomes evolved to combat host-encoded restriction systems. dZ-modified DNA can also reduce phage T5 exonuclease degradation, but has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from <i>Salmonella</i> phage PMBT28 (<i>purZ</i> (adenylosuccinate synthetase), <i>datZ</i> (dATP triphosphohydrolase), and <i>mazZ</i> ((d)GTP-specific diphosphohydrolase) were cloned into an <i>E. coli</i> plasmid, the level of dZ incorporation reached 19-20% of adenosine positions. dZ levels further increased to 29-44% with co-expression of a DNA polymerase gene from the same phage. High levels of dZ incorporation in recombinant plasmid are possible by co-expression of <i>purZ, mazZ, datZ</i> and phage DNA helicase, <i>dpoZ</i> (DNA polymerase) and <i>ssb</i> (single-stranded DNA binding protein SSB). This work expands our understanding of the dZ modification of DNA and opens new avenues for engineering restriction systems and therapeutic applications.