Fig 2: Real-time monitoring of recruitment of mKO1-RAD23B to local DNA damage. (a) Immunoblot analyses of endogenous and ectopically expressed XPC, DDB2, and RAD23B in the indicated cell lines. (b,c) Local DNA damage was induced in the indicated cell lines by three-photon absorption with an infrared femtosecond laser. Relative mKO1 intensities at damaged sites were quantitatively assessed over time. Inset in (b) shows enlargement of the graph in the early time range. The statistical significance assessed for the last time point is shown. *** P < 0.001. (d) Immunoblot analyses validating the effects of MLN4924. U2OS cells were pre-incubated for 2 h in the presence or absence of 1 µM MLN4924, and then irradiated with UVC at 10 J/m2 (or mock-irradiated) and further incubated for 1 h with or without MLN4924. Asterisk indicates non-specific reaction of the anti-XPC antibody. e The indicated cell lines were pre-treated for 2 h with or without 1 µM MLN4924, and recruitment of mKO1-RAD23B to local DNA damage was measured as in panels b and c. *** P < 0.001.

Fig 3: MG132-induced DDB2 accumulation depends on the proteasome subunit PSMD14. (a) Immunoblot analyses validating depletion of the proteasome subunits. U2OS cells were transfected with negative control siRNA (siNC) or siRNA targeting PSMD14 (siPSMD14), and cell lysates were prepared after incubation for the indicated number of days. All subsequent experiments were performed on the second day of post-transfection. (b,c) Cells expressing DDB2-mKO1 were treated with siNC or siPSMD14, and then incubated for 6 h in the presence or absence of 5 µM MG132. Based on live-cell images acquired with a confocal laser scanning microscope (b), percentages of cells with subnuclear DDB2 accumulation were measured as in Fig. 2d. (d) Immunoblot analyses validating expression of the XPC, DDB2, and PSMD14 proteins in the indicated cell lines. (e) Recruitment of PSMD14-mKO1 to local DNA damage was assessed quantitatively in the indicated cell lines. ** P < 0.01. (f) Representative live-cell images of PSMD14-mKO1 localization in cells that were mock-treated or treated for 6 h in the presence of 5 µM MG132 or 10 µM lactacystin.

Fig 4: Schematic representation of functional impacts of UPS on GG-NER. (a) Model for roles of UPS factors in the GG-NER process. The CRL4DDB2 E3 ligase bound to a DNA lesion ubiquitylates DDB2, XPC, and other proteins nearby (such as histones). Successful recruitment of XPC-RAD23 competitively suppresses ubiquitylation and degradation of DDB2. UPS factors such as the proteasome, RAD23, and p97/VCP are then recruited to degrade poly-ubiquitylated substrates. (b) Impacts of proteasome dysfunctions revealed by this study.

Fig 5: DNA-binding interactions of other DNA repair proteins. (A) The structure of eGFP-XPC (PDB codes 6CFI of Rad4 the yeast homolog to XPC and 4EUL). (B) A cartoon depiction of the DNA substrate used for XPC binding characterization, with UV damage sites shown in yellow and XPC binding shown in blue. Also shown is an example kymograph of eGFP-XPC binding and diffusing along the DNA in yellow. (C) CRTD analysis of XPC binding DNA with UV damage. (D) Distribution of motile and nonmotile XPC events. (E) An example MSD plot for analyzing XPC diffusion on DNA (D, in µm2/s). (F) Diffusion and a values for the diffusion of XPC on DNA. Event marked with asterisk was too short to determine an a value so it was defined as 1.0. (G) A structural model of APE1-tGFP from PDB code (5WNO and 4EUL). (H) Schematic and example kymograph of APE1 binding to DNA with nicks. (I) CRTD analysis of APE1 binding nicked DNA, with fit shown in blue. (J) A structural model of polß-tGFP, taken from PDB codes (4KLO and 4EUL) and the tGFP modeled in. (K) Example schematics of polß binding DNA containing nicks as well as a corresponding kymograph of an observation of polß binding. (L) CRTD analysis of polß binding nicked DNA, with the fit shown in blue. See Supplementary Figure S4.