N7004

The following compounds were used at the indicated final concentrations: PJ-34 (10 μM, ALX-270-289-M001, Enzo Life Science), Olaparib (1-10 μM, AZD-2281, Selleckchem), Rucaparib (1-8 μM, AG-014699, Selleckchem), Talazoparib (6.25-50 nM, BMN673, Selleckchem), β-Nicotinamide adenine dinucleotide hydrate (20-200 μM, N7004, Sigma-Aldrich), hydrogen peroxide (0.02-0.5 mM, H3420, Sigma-Aldrich), Methyl methanesulfunate, MMS (0.01%, 129925, Sigma-Aldrich), PARG inhibitor (10 μM, PDD00017273, Tocris), FK866 (1 μM, F8557, Sigma-Aldrich), MG132 (10 μM, M7449, Sigma-Aldrich), cycloheximide (50 μM, C7698, Sigma-Aldrich).

Crystals of S1_NAD and S1_E129D bound to NAD⁺ (Sigma-Aldrich N7004) were obtained by incubating protein at 8 to 10 mg/ml with 1 mM NAD⁺ for 10 min before setting up as hanging drops in a 1:1 ratio over a reservoir of 25% PEG 3350, 0.2 M potassium iodide. Crystal clusters grew in 6 to 12 days and were crushed to use as seeds to obtain single crystals. Crystals were transferred into cryoprotectant consisting of 25% v/v glycerol prior to plunging into liquid nitrogen. To obtain S1_ADPR/NA crystals, the S1(C41S) was complexed with 1 mM NAD+ and crystallized over the physiologically permissive solution 0.5 M (NH₄)₂SO₄ and 0.1 M Hepes, pH 7. Cryoprotectant consisted of an additional 25% v/v glycerol, added to the reservoir. The structure of S1_AB was obtained when S1(C41S) was co-complexed with both 1 mM NAD⁺ and 1 mM 3AB. Crystals were grown in 0.2 M LiCl and 14% w/v PEG 3350 and transferred to a cryoprotectant solution with an additional 27% v/v glycerol prior to data collection. The structure of S1_PJ34 was obtained when S1(C41S) was co-complexed 1 mM PJ34 and crystallized over a reservoir containing 0.15 M NaCl, 0.1 M Tris(hydroxymethyl)aminomethane, pH 8.0, and 8% w/v PEG 8000. Crystals were soaked into a solution with an additional 25% v/v glycerol before flash freezing in liquid nitrogen.