Tempest liquid handler

All TR-FRET assays were ran in Black 384-well ProxiPlate Plus (Perkin-Elmer, USA), in a buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, 0.5 mM TCEP and 0.05% Tween 20, with a final assay volume of 10 μL. An Echo E5XX (Beckman Coulter, USA) acoustic liquid dispenser was used to create final concentration ranges from 90 μM down to 2.25 nM for DCD23. Final concentrations to 5 nM WT full length CRBN/DDB1 complex, 750 nM Sulfo-Cy5 fluorescent Aiolos-based peptidic probe (Figure S6C) (Cambridge Research Biochemicals, UK) and 0.5 nM MAb Anti-6HIS-Terbium cryptate Gold (Cisbio, France) were added with a Tempest liquid handler (Formulatrix, USA). Finally, degraders were added to a final concentration of 10 μM (or 1 μM for CC885 due to solubility issues) with the Echo. Plates were sealed, centrifuged at 200 g for 1 min and stored overnight at 4°C. The TR-FRET signals were read using a PHERAstar FSX plate reader (BMG Labtech, Germany). Final signal was measured as the ratio: $\mathrm{T}\mathrm{R}-\mathrm{F}\mathrm{R}\mathrm{E}\mathrm{T}\mathrm{s}\mathrm{i}\mathrm{g}\mathrm{n}\mathrm{a}\mathrm{l}=\frac{\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{l}1\left(665\mathrm{n}\mathrm{m}\right)}{\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{e}\mathrm{l}2\left(620\mathrm{n}\mathrm{m}\right)}$
Chanel1 at 665nm representing a positive FRET and chanel2 at 620nm representing the emission of the terbium tag when no FRET occurs (Figure S6C).
Kd values were used along with the TR-FRET IC₅₀ values to calculate the Ki using the Cheng-Prusoff equation: $\mathrm{K}\mathrm{i}=\mathrm{I}\mathrm{C}50/\left[1+\left(\mathrm{H}\mathrm{T}\mathrm{R}\mathrm{F}\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{e}\mathrm{d}\mathrm{l}\mathrm{i}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)/\mathrm{K}\mathrm{d}\right]$

To test whether the Polθ inhibitors were causing signal interference by intercalating DNA, ART558 was dispensed into 384-well black low volume non-binding plates (Greiner). For each compound, 100 nL of a 1.5-fold dilution series was dispensed starting from a 12 mM top concentration of compound in DMSO using the D300e digital dispenser (Tecan). A DMSO control was also included. Wells were normalised and backfilled with DMSO such that all wells contained 1% (v/v) after the addition of 10 μL of Polθ DNA product solution. 10 μL of a solution containing 200 nM Polθ product DNA in assay buffer (25 mM Tris-HCl pH 7.5, 12.5 mM NaCl, 0.5 mM MgCl₂, 5% (v/v) glycerol, 0.01% (v/v) Triton x-100, 0.01% (w/v) Bovine γ-Globulin, 1 mM dithiothreitol) and 5 μL of detection reagent (25 mM Tris-HCl pH 7.5, 10 mM EDTA, 2.5% (v/v) PicoGreen) were dispensed separately using a Tempest liquid handler (Formulatrix). Plates were then read on the CLARIOstar Plus (BMG Labtech) using the settings described above. Two known DNA intercalators: mitoxantrone and doxorubicin were used as positive controls. IC₅₀ data were analysed using GraphPad Prism V.8.4.2 (GraphPad Software Inc, San Diego, CA).