In vitro ADPr assays
A variety of in vitro ADPr assays were used to measure the ability of enzymes to modify or demodify different substrates.

Recombinant proteins and peptides
Recombinant proteins are purified as described previously (Langelier et al., 2011, Langelier et al., 2014, Gibbs-Seymour et al., 2016, Fontana et al., 2017, Dunstan et al., 2012). Peptides were purchased from EpiCypher or custom made. Nucleosomes were from EpiCypher.

Enzymatic preparation of the modified histone peptides
Recombinant Dim-5 (the homolog of human SUV39H1/2) and SIRT2 were purified as previously described (Rack et al., 2014, Zhang et al., 2002). Recombinant p300 was purchased from Enzo Life Sciences. HDAC2 was purchased from Active Motif. For histone phosphorylation reactions we used the activated Aurora B fragment called Baronase, which was a gift from the Barr lab (Nunes Bastos et al., 2013). H3 peptides were purchased from EpiCypher. H3 peptides (either WT or Ser-ADPr modified as described above) were incubated in either; HAT buffer (p300) - 50 mM Tris-HCl pH 8.0, 1mM DTT, 100 μM Acetyl-CoA, 10% glycerol for 30 min at 30°C; phosphorylation buffer (Baronase) - 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 10 mM DTT for 60 min at 37°C; methyltransferase buffer (Dim-5,) – 50mM Glycine pH 9.8, 2 mM DTT, 10% glycerol for 20 min at room temperature. Reactions were then analyzed by SDS-PAGE and western blotting (detailed below). HDACi reactions (HDAC2, SIRT2) were performed in reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2, which were subsequently supplemented by PARP1/HPF1, activated DNA and 50 μM NAD+ spiked with 32PNAD+. The modification reaction proceeded at room temperature for 20 min before addition of the PARPi Olaparib at 1 μM. Reactions were then analyzed by SDS-PAGE and autoradiography.

Standard radioactivity-based ADPr assay
Recombinant proteins or peptides were ADPr by PARP1 in the presence or absence of HPF1 and histone peptides. PARP1 concentration in the assays was 1 μM unless stated otherwise, HPF1 was always equimolar to PARP1, histone peptides were used at 0.5 μg per reaction, and recombinant nucleosomes were at 1 μM. The PARP reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2, activated DNA and 50 μM NAD+ spiked with 32PNAD+. The modification reaction proceeded at room temperature for 20 min before addition of the PARPi Olaparib at 1 μM. Reactions were then analyzed by SDS-PAGE and autoradiography.

Inverted polarity native gels for ADPr detection
This simple, non-radioactive method allows visualization of both substrates and products of ADPr reactions. ADPr reactions were performed as described above, except with 2 μg histone peptide per reaction, and in the presence of non-radioactive NAD+. Samples were mixed with TBE Sample Buffer and loaded on 20% TBE gels in TBE Running Buffer. Samples were run with inverted polarity at 200 V for 1 hr. Gels were then fixed for 30 min in 10% glutaraldehyde, washed in H20 for 3 × 10 min, then stained with Imperial Protein Stain for 1 hr.

In vitro ADP-ribosyl glycohydrolase assays
The assays were performed as in Fontana et al., 2017. Briefly, H3 peptides were incubated with PARP1 and HPF1, under the conditions described above, and stopped by addition of Olaparib. ARH3 was then added to the reactions for incubation at room temperature for 30 min. Reactions were then analyzed by SDS-PAGE and autoradiography. ARH3 concentration was at 1 μM.