Fig 1. Biochemical analysis of Vpr_mus-induced CRL4^DCAF1 specificity redirection.

(A-C) GF analysis of in vitro reconstitution of protein complexes containing DDB1/DCAF1-CtD, Vpr_mus and SAMHD1 (A), SAMHD1-ΔCtD (B) or T4L-SAMHD1-CtD (C). Elution volumes of protein molecular weight standards are indicated above the chromatogram in A. Coomassie blue-stained SDS-PAGE analyses of fractions collected during the GF runs are shown below the chromatograms, with boxes colour-coded with respect to the chromatograms. SAM–sterile α-motif domain, HD–histidine-aspartate domain, T4L –T4 Lysozyme. The asterisk and double asterisk indicate slight contaminations with remaining GST-3C protease and the GST purification tag, respectively. (D-G) In vitro ubiquitylation reactions with purified protein components in the absence (D) or presence (E-G) of Vpr_mus, with the indicated SAMHD1 constructs as substrate. Reactions were stopped after the indicated times, separated on SDS-PAGE and visualised by Coomassie blue staining.