Fig. 1
From: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
USP48 specifically deubiquitinates H2ABRCA1ub. a Schematics of the fluorescence polarization screen. Recombinant nucleosome core particles are site specifically ubiquitinated using the E3 ligase named and TAMRAUb. Upon addition of a DUB, cleavage can be followed by a decrease in FP signal. b USP48 prefers nucleosomal substrates. Site-specific cleavage of H2A168ub, H2APRC1ub, and H2ABRCA1ub in relation to activity on minimal substrate. Observed k-values from fitting the raw data of the FP assay (kobs(NCP)) (Supplementary Fig. 1c) were normalized to observed k-values on minimal substrate (kobs(Rho)) obtained from fitting an exponential function to the traces in Supplementary Fig. 1e). A value above one indicates that NCPs are the preferred substrate. The mean of two technical replicates ± SEM is shown. c Time course of USP48 cleavage of H2A168ub, H2APRC1ub, and H2ABRCA1ub, anti-H2A western blotting. USP48 only cleaves efficiently when more than one ubiquitin is present on the BRCA1 site. The blot shown is representative of two experiments. d USP48 cleaves multi-monoubiquitinated H2ABRCA1ub faster than multi-monoubiquitinated H2A168ub and H2APRC1ub. Quantification of the initial reaction velocity (V0) of USP48 measured by the liberation of free TAMRAUb. Gels used for quantification are shown in Supplementary Fig. 2a. The mean of two technical replicates ±SEM is shown. e USP48 does not cleave H2APRC1ub when H2ABRCA1ub is present, but all H2ABRCA1ub is cleaved. Left panel: cleavage of NCPs, ubiquitinated on PRC1 site with unlabeled ubiquitin, and on the BRCA1 site with TAMRAub by USP48 was followed on gel. The TAMRA fluorescence is used as readout. Right panel: quantification of the initial reaction velocity (V0) measured by liberation of free ubiquitin. The mean of two technical replicates ± SEM is shown
