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.