Fig. 4: AdfN^T4 is a NADAR protein with DNA ADP-ribosylglycohydrolase activity.

a Bacterial 2-hybrid experiment measuring the association of catalytically inactive DarT1 (DarT1*) bait with the indicated prey proteins, as in Fig. 1c. Dark blue color indicates an association between the proteins; white means they do not associate. Representative image of three independent experiments is presented. b AlphaFold2 model of AdfN^T4, indicating a NADAR fold with conserved catalytic residues in orange, and the helix missing from AdfN^RB69 in red. c Representative plaque assay of RB69 in the presence of DarTG1 with ectopic expression of the indicated adfN^T4 variant. See Fig. S5 for quantification of the titer and plaque sizes of four replicates. d ADP-ribosylation of DNA measured using a dot blot following an in vitro incubation with either AdfN^T4 or AdfN^T4(E36A). Representative image of three independent experiments is presented. e Colony forming units (c.f.u.s) of bacteria expressing darT1 and either bearing an empty vector, a vector expressing darG1, or adfN^T4. DarT1 was repressed with glucose or induced with arabinose as indicated. f Plaque assays of the indicated phage in the presence or absence of darTG1, with empty vector or expressing adfN^T4 from a plasmid. Representative image of three independent experiments is presented. Source data are provided as a Source Data file.