Anti h2a

Calf thymus histone-binding assays were performed by incubating 50 µg of calf thymus histones (Worthington) with 10 µg of recombinant GST fusion proteins in buffer (50 mM Tris at pH 7.5, 1 M NaCl, 1% NP-40) overnight. GST fusion protein histone interactions were assessed by adding glutathione sepharose 4B (GE) beads for 1 h and washing five times in buffer. The beads were resuspended in 5× SDS sample buffer, run on a 4%–12% Tris-Bis gel, and transferred to a nitrocellulose membrane followed by Western blot analysis. We used the following antibodies: anti-H3 (1:5000; Active Motif, 39163), anti-H2A (1:2000; Millipore, 07-146), and anti-H2B (1:2500; Millipore, 07-371).

For the single turnover assays, the E2 was pre-charged with ubiquitin. Concentrations are given for Figs 2c and 5, in brackets for Fig. 5 and Supplementary Fig. 3. To load the E2, E1 at 0.5 μM was mixed with 100 μM (70 μM) UbcH5c in the presence of 100 μM (70 μM) ubiquitin Mg²⁺ and 3 mM ATP in buffer, 50 mM Tris/HCl (pH 7.5), 100 mM NaCl and 10 mM MgCl₂. The sample was incubated for 30–60 min at room temperature. The reaction was stopped with 50 mM EDTA final concentration and incubated on ice for 2 min. This mixture was then used for the discharge reactions, a final concentration of 15 μM (20 μM) E2~Ub was incubated at 32 °C with 100 nM (1 μM) of RNF168 RING domain alone or in the presence of 10 μM (5 μM) NCPs or 20 μM (5 μM) dimers. Samples were stopped at given times and were analysed by Coomassie staining on SDS–PAGE in non-reducing conditions and subsequently western blot analysis was carried out using anti-H2A (Millipore, 07–146) or anti-Ub (Santa Cruz, P4D1) antibodies. Samples were run on either 12% or 4–12% NuPAGE gels in MOPS buffer (Life Technologies). Images were taken using ChemiDoc XRS system (BioRad) and ImageLab programme. Quantifications were done using the peak height value of the E2~Ub band on Coomassie gels in ImageLab and GraphPad was used to prepare graphs.