Fig. 2
From: USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A
USP48 cleavage rates on H2ABRCA1ub are modulated by an auxiliary ubiquitin. a An auxiliary ubiquitin activates USP48 processivity. Gel-based assay to measure USP48 cleavage of H2ABRCA1ub. TAMRAUb was used as readout. Cleavage was recorded under several different substrate and enzyme concentrations. Four (stacked) gels are shown as an example (Supplementary Fig. 3 for all gels). b Quantification of (a. Four out of 11 different conditions shown as an example (Supplementary Fig. 3 for full panel). Solid lines show fit obtained by global fitting of all 11 quantified time courses and binding data to the model defined in e using Kintek Explorer. c USP48 binds with similar affinities to ubiquitinated and unmodified NCP. USP48 is titrated to 50 nM of unmodified NCP or NCPBRCA1ub and analyzed by native gel-shift assays. A Kd of 1 µM is estimated. d USP48 binds nucleosomes of different ubiquitination status with the same affinities. Binding of USP48 to H2ABRCA1ub, H2ABRCA1ub1, and unmodified nucleosomes measured by surface plasmon resonance. Nucleosomes were immobilized on the surface. Normalized RU values of 10 successive injections of different USP48 concentrations are fitted by a one-phase binding model using GraphPad Prism (raw data in Supplementary Fig. 3b). Kd and SE of the fit are indicated. e USP48 cleaves H2ABRCA1ub in nucleosomes 50–150 times faster than when auxiliary ubiquitin is present. Kinetic model describing USP48’s cleavage pattern on H2ABRCA1ub with the fitted values for kcat(ub3), kcat(ub2), and kcat(ub1), and the SE of the fit. f Parameters for kcat(ub3), kcat(ub2), and kcat(ub1) in e are well constrained by the data. Evaluation of the goodness of fit. The upper three panels show how well defined the lower and upper boundaries are for the individual parameters. kcat(ub1) and kcat(ub2) fall into a well-defined local χ2 minimum. For kcat(ub3), the lower boundaries are well defined which allows the conclusion that Kcat(ub3) should always be faster than kcat(ub1). The lower panel shows how χ2 varies when two of the fitted variables are varied against each other. Red indicates a χ2 minimum
