Uncropped gel—nuclease data from: A nicking class 1 OLD family nuclease encoded by <em>Vibrio cholerae</em> inhibits virbiophage replication and is countered by a direct inhibitor

Bacteria are constantly threatened by their viral predators (phages), which has resulted in the development of defense systems for bacterial survival. One family of defense systems found widely across bacteria are OLD (for overcoming lysogenization defect) family nucleases. Despite recent discoveries regarding Class 2 and 4 OLD family nucleases and how phages overcome them, Class 1 OLD family nucleases warrant further study, as there has only been one anti-phage Class 1 OLD family nuclease described to date. Here, we identify the first bacteria-encoded Class 1 OLD family nuclease that exerts anti-phage activity: the Vibrio cholerae encoded Class 1 OLD family nuclease Vc OLD. &nbsp;We describe its disruption of the genome replication of the lytic vibriophage ICP1. Furthermore, we examine its in vitro activity, identifying Vc OLD as a DNA nickase. Finally, we identify the first direct inhibitor of a Class 1 OLD family nuclease, the ICP1-encoded Oad1. Our research further illuminates Class..., Nuclease assays with purified Vc OLD and Oad1 and bovine serum albumin (BSA) (Fisher BP1600) were carried out in 50-mM Tris–HCl, pH&nbsp;=&nbsp;7.4, 50-mM NaCl, 10-mM MgCl2, 10-mM MnCl2, 10-mM CaCl2, 10NiCl2, and 10-mM ZnCl2. Frozen samples of all proteins were thawed and added to the buffer at varying concentrations. All nucleic acid substrates were eluted into H2O after nucleic acid collection. Double-stranded DNA (dsDNA) substrate was extracted from bacteria or phage following the procedure outlined in “Post-infection deep sequencing and coverage mapping.” Similarly, single-stranded DNA (ssDNA) (from M13 phage) was ordered from New England Biolabs (N4040S), extracted, and eluted into water. Total RNA was prepped from V. cholerae E7946 as described previously. Briefly, Bacterial strains were grown to an OD600&nbsp;=&nbsp;0.3, collected, and mixed 1:1 with ice-cold methanol. Methanol-treated samples were pelleted at 7000 × g at 4°C, and the resulting pellets were washed in ice-cold 1× ..., # Uncropped gel—nuclease data from: A nicking class 1 OLD family nuclease encoded by *Vibrio cholerae* inhibits virbiophage replication and is countered by a direct inhibitor Dataset DOI: [10.5061/dryad.rxwdbrvnt](10.5061/dryad.rxwdbrvnt) ## Description of the data and file structure ### Files and variables #### File: Figure_S15A-_Replicate_B.tif **Description:** These data are linked to Supplemental Figure 15 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, the lanes are: Plasmid incubated with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 100nM Vc OLD and 2nM BSA, plasmid incubated with 100nM Vc OLD and 4nM BSA, plasmid incubated with 100nM Vc OLD and 100nM BSA, plasmid incubated with 100nM Vc OLD and 2500nM BSA, plasmid incubated with 100nM Vc OLD and 5000nM BSA, plasmid incubated with 5000nM BSA and without Vc OLD, 100nM Vc OLD incubated without plasmid, ...,