Fig 1: Biochemical characterization of PARP9:DTX3L catalysed NAD+ ubiquitylation. (A) Schematic diagrams showing the proposed DELTEX product in different studies. Left panel: ADP-ribosylated Ub; Right panel: ubiquitylated NAD+. (B) Hydrolysis of DTX3L RD catalysed Ub-NAD+. Following one reaction performed with DTX3L RD in (B), the indicated ADPr hydrolases and DUBs were added and further incubated. The samples were analyzed on an SDS-PAGE gel, which is then visualized by Coomassie staining and autoradiography. The arrows indicate various hydrolases. The arrows indicate various hydrolases. (C) Schematic diagram showing the NUDT16 cleavage specificity of ADP-ribosylated product and DELTEX product. NUDT16 treatment reverses radioactivity from 32P ADP-ribosylated PARP1 E988Q but not the 32P Ub-NAD+. (D) PARP9:DTX3L and DELTEX E3s ubiquitylate NAD+ on the adenine-proximal ribose. 32P Ub-NAD+ was generated by incubation of indicated E3s with E1, E2, ATP, Ub and NAD+ which is spiked with 32P NAD+, and next treated with NUDT16 or NH2OH. 32P ADP-ribosylated PARP1 E988Q was treated with NUDT16 to serve as control. The samples were analyzed on an SDS-PAGE gel, followed by Coomassie staining and autoradiography. NUDT16 removed radioactivity from PARP1 E988Q auto-modification but had no effect on the 32P Ub-NAD+ adduct, confirming that Ub is attached to the adenine-proximal ribose.
Fig 2: DTX E3s catalyse conjugation of Ub to the 3’ hydroxyl of NAD+/ADP-ribose(A) Schematic diagram of expected NUDT16 cleavage specificity. NUDT16 treatment eliminates radioactivity from a 32P-NAD+-labelled protein if it is attached to the dinucleotide via C1" but not when it is attached to the adenine-proximal ribose ring.(B) Poly(ADP-ribosyl)ated PARP1 WT, mono(ADP-ribosyl)ated PARP1 E988Q, and the DTX product were created by incubating the relevant components with NAD+ and then treated or not with NUDT16. The samples were analysed on an SDS-PAGE gel, which is then visualised by Coomassie staining and autoradiography. NUDT16 reverses PARP1/PARP1 E988Q auto-modification but has no effect on the DTX2-catalysed Ub-NAD+ adduct, consistent with the adenine-proximal Ub attachment.(C) Flowchart of Gly-Gly-ADP-ribose generation. The DTX reaction followed by trypsin digestion results in ADP-ribose attached to the tryptic Ub remnant Gly-Gly-. Trypsin cleavage specificity is indicated.(D) Chemical formulas of Gly-Gly-ADP-ribose and reference molecules used in NMR.(E) NMR localisation of the Gly-Gly remnant to the 3’ hydroxyl group of the proximal ribose of ADP-ribose based on the largest shifts (Δppm) in δ 1H and 13C values in these positions. For ADP-ribose and Gly-Gly-ADP-ribose, δ 1H and 13C values are provided only for the major β anomer (full data and explanation in Supporting information).
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