Figure 4. UVRAG Antagonizes CAND1 and Promotes the Ubiquitin-mediated Proteolysis of CRL4^DDB2 E3 Ligase In GG-NER in vivo.

(A) Effect of UVRAG on UV-induced Cul4A neddylation and Cul4A–CAND1 interaction. A375 cell lines stably expressing control shRNA, or expressing UVRAG-specific shRNA complemented with empty vector, WT UVRAG, or with its DDB1-binding defective L286F mutant were UV-C treated. WCL were immunoprecipitated with anti-Cul4A followed by IB with anti-Cul4A or anti-CAND1 antibody.

(B) Densitometric quantification of the neddylated Cul4A/un-neddylated Cul4A ratio under the indicated condition in (A). Data shown represent mean ± SD from three independent experiments. n.s., not significant.

(C) UVRAG and CAND1 bind to Cul4A in a mutually exclusive manner. WCL prepared from A375 cells were immunoprecipitated with control IgG, anti-CAND1, anti-Cul4A, anti-UVRAG, or anti-DDB1, followed by IB with the indicated antibodies.

(D) UVRAG antagonizes the inhibitory effect of CAND1 on Cul4A neddylation. A375 cells transiently transfected with different amounts of Flag-UVRAG and/or CAND1 were UV-C treated. WCL were immunoprecipitated anti-Cul4A antibody followed by IB with anti-Nedd8 antibody.

(E) UVRAG antagonizes the inhibitory effect of CAND1 on the DDB1-Cul4A interaction. A375 cells transiently transfected with Flag-UVRAG and/or myc-CAND1 were UV-C treated. WCL were immunoprecipitated with control or anti-Cul4A antibody followed by IB with anti-DDB1 and Cul4A antibodies.

(F) Effect of UVRAG on UV-induced histones and XPC ubiquitination, and DDB2 degradation. A375 cell lines stably expressing control shRNA, or expressing UVRAG-specific shRNA complemented with empty vector, WT UVRAG, or with its DDB1-binding defective L286F mutant were treated with UV-C irradiation. WCL were immunoblotted for histone H3/H4 ubiquitination, XPC ubiquitination, and DDB2 degradation.

(G) UVRAG antagonizes CAND1-mediated inhibition of DDB2 ubiquitination. 293T cells were transfected with Myc-DDB2 and HA-ubiquitin (Ub), along with different amount of CAND1-V5 and/or Flag-UVRAG. At 48 hr post-transfection, cells were UV-irradiated and treated with MG132. WCL were used for IP with anti-Myc followed by IB with anti-HA.

(H, I) UVRAG deficiency impedes UV-induced DDB2 degradation and CPD repair. A375 cells stably expressing control shRNA or UVRAG shRNA were UV-C treated for 30 min and let recovered for a period of time as indicated. DDB2 levels and UV-induced CPD damage were determined by immunostaining with anti-DDB2 and anti-CPD, respectively (H). The retention of DDB2 at CPD foci is quantified (I). Scale bar, 10 µm. ****p < 0.0001; n.s., not significant.

See also Figure S6 for additional information.