DNA glycosylases provide antiviral defence in prokaryotes

. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence datawith the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication.

Each clone within the library houses a cosmid with a soil DNA insert, which carries genes from soil-derived microorganisms. Soil-derived genes can therefore be expressed heterologously in our library system. We performed our functional screen using the coliphage T4. To grow up libraries, we scraped frozen library stocks ofEC100 carrying megapools 3–16 of the AZ52 DNA library into separate tubes with 10 ml LB supplemented with 12.

For mass spectrometry, purified oligonucleotide samples were dried using vacuum centrifugation and dissolved in 50/50 water/acetonitrile with 0.001% triethylammonium bicarbonate. The pH of the solution was found to be comparable to that of deionized water. The samples were introduced to the mass spectrometer by manual injection using a Hamilton syringe applying pressure by hand at approximately 10 μl min.

The authors thank all members of the Marraffini laboratory for helpful discussion and encouragement; B. R. Levin for providing the λvir, T4 and T7 phages; A. Harms for providing the BASEL phage collection; the Coli Genetic Stock Center at Yale University forACT-01 strain; A. Z. Fire for the pPD207.846 plasmid; A. J. Meeske for the pAM39 plasmid; P. Maguin for laying the framework forgene neighbourhood analysis; A. J. Varble and P. M. Nussenzweig for experiment suggestions; C. G.

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