Fig 1: UVR8 does not bind to nucleosomes in vitro. a Purified recombinant RCC1 and UVR8 (- and + UV-B treatment) were electrophoretically separated on SDS-PAGE together or in the absence of reconstituted nucleosomes (Nucl.) and stained by Coomassie Blue. Samples were boiled or non-boiled before loading the gel. b Electrop horetic mobility gel shift assay of nucleosome binding by RCC1 and UVR8. Between 0.15 - 10 µM RCC1 and 0.15 - 40 µM UVR8 (- and + UV-B treatment) were added to reconstituted mononucleosomes. Binding was assayed by EMSA and visualized by ethidium bromide staining
Fig 2: Structural comparison of UVR8 with the nucleosome binding interface of RCC1 shows that UVR8 lacks positive surfaces to interact with nucleosomes. a Overview of the Drosophila RCC1-nucleosome complex determined by X-ray crystallography [21]. Both RCC1 molecules bind identically to the nucleosome. b DmRCC1 viewed through the nucleosome shows the nucleosome interaction interface. Left: For RCC1, the solvent accessible surface is shown coloured in terms of electrostatic potential [52, 53]. The nucleosome is depicted as semi-transparent. Right: The same representation of RCC1 with the nucleosome contact surface outlined in pink. DNA and histone-binding regions are indicated. c, d, e Comparison of electrostatic surface potential of HsRCC1 (pdb 1A12) (c), ScSMR1/Prp20 (pdb 3OF7) (d) and UVR8 (pdb 4DNW) (e, upper) with an orientation identical to that of DmRCC1 in (b). The electrostatic surface potential for the opposite side of UVR8 is also shown by rotation of the molecule (e, lower). PyMOL (http://www.pymol.org) and APBS plugin for PyMOL (MG Lerner and HA Carlson, 2006, University of Michigan, Ann Arbor) was used for visualization of these structures. f Structure-based multiple sequence alignment of RCC1 homologs with UVR8. Shaded residues indicate those confirmed or proposed to be critical for RCC1 binding to the nucleosome
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