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. 2017 Jun 26;6:e28533. doi: 10.7554/eLife.28533

Figure 2. ARH3 hydrolyses Ser-ADPr.

(A) Glycohydrolysis of mono-ADP-ribosylated H3 histone peptide (aa 1-21). ADP-ribosylated H3 peptide was obtained by incubation with PARP1 and HPF1 using radioactively labelled NAD+ as an ADPr donor. After stopping the modification reaction with PARP inhibitor, the indicated glycohydrolases and related proteins were added to the substrate as shown on the figure. Lower panel – Coomassie stained gel of the proteins used in the reaction that also serves as a loading control. (B) Removal of poly-ADP-ribosylation from the recombinant histone H1 substrate by various glycohydrolases. Reaction performed as described in A. (C) Comparison of the hydrolase reactions on the poly- and mono-ADP-ribosylated histone H1 substrates. Lower panel – Coomassie stained gel of the proteins used in the reaction that also serves as a loading control. (D) Schematic representation of the SILAC-based strategy to quantify ADPr removal upon in vitro incubation of purified Ser-ADPr histones with or without recombinant ARH3 (top panels). Log2 of summed peptide intensities were plotted against log2 Heavy/Light SILAC ratios for each condition (bottom panels). ADPr peptides are colored in red. (E) Autoradiogram analysis of Ser mono-ADPr hydrolysis with a non-histone protein substrate. The ADP-ribosylated automodification domain of PARP1 (aa 374-525) was used in this assay and the reactions were supplemented with the indicated hydrolases. In panel panels A, B, C and E, the reaction products were separated by SDS-PAGE and analysed by autoradiography. The signals relating to the specific ADP-ribosylated protein are indicated in each panel.

DOI: http://dx.doi.org/10.7554/eLife.28533.006

Figure 2—source data 1. MaxQuant evidence table related to Figure 2D.
ADPr identified peptide features from SILAC experiments performed to quantify ADPr removal upon in vitro incubation of purified Ser-ADPr histones with or without recombinant ARH3 (Figure 2D).
DOI: http://dx.doi.org/10.7554/eLife.28533.007
elife-28533-fig2-data1.xlsx (54KB, xlsx)
DOI: 10.7554/eLife.28533.007

Figure 2.

Figure 2—figure supplement 1. PARP1 E988Q generates mono Ser-ADPr on itself and on substrates.

Figure 2—figure supplement 1.

(A) Removal of poly-ADP-ribosylation from the recombinant histone H1 substrate by the glycohydrolases PARG, ARH3, PARG and ARH3 together, and TARG1 as indicated. ADP-ribosylated histone H1 was obtained by incubation with PARP1 and HPF1 using radioactively labelled NAD+ as an ADPr donor. After stopping the modification reaction with PARP inhibitor, the indicated glycohydrolases were added to the substrate as shown on the figure. (B) High-resolution ETD fragmentation spectrum of a PARP1 E988Q peptide modified by ADP-ribose on Ser499. (C) High-resolution ETD fragmentation spectrum of a H1.0 peptide modified by ADP-ribose on Ser103.
DOI: http://dx.doi.org/10.7554/eLife.28533.008