FIGURE 2.

C1orf124 interacts and co-localizes with PCNA at UV light-induced damage sites. A, TAP was performed using 293T cells stably expressing SFB-tagged C1orf124. The results from mass spectrometry analysis are shown. B, sequence alignment of the PIP box motif of C1orf124 with conserved PIP box motifs of other PCNA-interacting proteins, namely FEN1, p21, and XPG. The consensus PIP box sequence (Qxxψxxθθ, where ψ = L/V/I/M and θ = Y/F) is indicated. C, C1orf124 interacts with PCNA via the PIP box motif. 293T cells were transfected with plasmid encoding Myc-tagged PCNA. Cell lysates were incubated with GST, GST-C1orf124ΔPIP, or GST-C1orf124, and immunoblotting was performed using the indicated antibodies. WB, Western blot. D, PCNA interacts with C1orf124 in the absence and presence of UV damage. 293T cells were cotransfected with plasmids encoding SFB-tagged PCNA and Myc-tagged C1orf124. 24 h later, cells were left untreated or treated with 100 J/m² UV-C light and collected 4 h later. Coprecipitation was carried out using S-protein beads, and immunoblotting was performed using the indicated antibodies. E, the UBZ domain of C1orf124 is required to bind to ubiquitinated PCNA. 293T cells were transfected with plasmids encoding SFB-tagged C1orf124 and C1orf124ΔUBZ. 24 h later, cells were treated with 100 J/m² UV-C light and collected 4 h later. Coprecipitation was carried out using S-protein beads, and immunoblotting was performed using the indicated antibodies. F and G, the PIP box motif and UBZ domain mediate the recruitment of C1orf124 to DNA damage sites. HeLa cells were transfected with constructs encoding SFB-tagged full-length (FL) C1orf124, C1orf124ΔSHP, C1orf124ΔPIP, or C1orf124ΔUBZ. Cells were treated with laser micro-irradiation (F) or 10 J/m² UV-C light (G) and incubated for 6 h prior to immunostaining with the indicated antibodies. RPA, replication protein A.