Figure 3 |. Conformational changes on ubiquitination.

a, Non-ubiquitinated ID (cyan, salmon and blue for FANCI, FANCD2 and DNA, respectively) superimposed on ID^Ub (cyan, magenta and yellow for FANCI^Ub, FANCD2^Ub, and DNA, respectively) by aligning FANCI. Ub^D2 is red (Ub^I obscured in this view). The rotation axis is shown as a thick black stick with the rotation indicated by a circular arrow. FANCD2 movement is indicated by a dashed arrow linking equivalent structural elements. The CTD-CTD interaction region is labeled “zipper”. Orientation similar to Fig. 2a. b, View looking approximately down the vertical axis of a with the two complexes rendered as semi-transparent surfaces to reveal the DNA inside. c, FANCI (left top, cyan), FANCI^Ub (left bottom, cyan), FANCD2 (right top, pink), and FANCD2^Ub (right bottom, pink) proteins showing residues (thick sticks) with a reduction in solvent accessibility due to interactions between FANCI and FANCD2 (top pair, blue sticks), between FANCI^Ub and FANCD2^Ub (bottom pair, red sticks), and between each protein and the ubiquitin of the other paralog (bottom pair, green sticks). Yellow spheres mark ubiquitination sites. d, Close-up view of the ID and ID^Ub complexes superimposed on the FANCD2 ubiquitination site (top) or on the FANCI ubiquitination site (bottom). Non-ubiquitinated ID is transparent gray. e, The ID^Ub CTD zipper looking down the vertical axis of b. Residues unstructured in non-ubiquitinated ID are shown in darker shades and are labeled. Dashed cyan line is a loop unstructured in both complexes. f, SDS-PAGE gel of the de-ubiquitination of wild type ID^Ub and mutant I^mutD^Ub complexes, both at 940 nM, by USP1-UAF1 (400 nM) at the indicated time points, detected by Coomassie staining (top) or anti-ubiquitin immunoblot (bottom). The positions of the substrates and products are marked. Repeated n=3 times.