Extended Data Fig. 10: USP3 does not interact directly with Poly-ADP-ribose chains.
From: XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair
a, Cartoon of GFP-tagged USP3 constructs employed in this work. b, Representative ScanR images of chromatin retention of USP3FL-GFP and USP3ZnF-GFP in WT and XRCC1−/− U2OS cells, following mock-treatment or for 4 h with 0.1 mg/ml MMS in the presence/absence of 10 μM PARPi as indicated. Cells were transfected with H2B–mCherry and USP3–GFP constructs 24 h before experiment. Cells were fixed with PFA to show total protein levels or were pre-extracted with detergent prior to fixation to show chromatin-bound proteins. c, Quantification of experiments depicted in b. Data are means (±s.e.m.) of three independent experiments, and statistically significant differences were determined by two-way ANOVA with Sidak’s multiple comparisons test (p values are indicated). d, Immunoblot of PARP1 and USP3ZnF-GFP levels in anti-ADP-ribose immunoprecipitates from WT and XRCC1−/− U2OS cell extracts following mock treatment or 1 h after treatment with 1 mM H2O2 for 20 min. A representative blot from one of two independent experiments is shown. e, Coomassie staining of purified recombinant human USP3 employed in the ADP-ribose binding assay shown in f. Single purification. f, Binding of full length USP3 and XRCC1161–406 (positive control) to calf thymus histones mock-ribosylated (-NAD+) or ADP-ribosylated (50 μM NAD+) with recombinant PARP1. Data are means (±s.e.m.) of three independent experiments.
