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

## TL;DR

This paper explores how a modified DNA base called dZ helps phage DNA resist bacterial restriction systems and shows how to produce dZ-modified DNA in E. coli.

## Contribution

The study demonstrates high dZ modification levels in recombinant plasmids through co-expression of phage-derived genes.

## Key findings

- 53% of tested restriction enzymes are resistant or partially resistant to dZ-modified DNA.
- dZ-modified DNA can resist phage T5 exonuclease degradation but not RecBCD digestion.
- Co-expression of phage genes in E. coli achieves up to 44% dZ modification in plasmid DNA.

## Abstract

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 Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into an E. coli 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 purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (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.