Dissociation of CAK from Core TFIIH Reveals a Functional Link between XP-G/CS and the TFIIH Disassembly State
Figure 1
In vivo recruitment of holo TFIIH to DNA damage sites in NER-proficient HeLa cells.
(A) Holo TFIIH is recruited to localized DNA damage sites. HeLa cells were grown on coverslips, irradiated with 100 J/m2 UV through a 5 µm isopore polycarbonate filter, cultured for 0.5 h and then fixed with 2% paraformaldehyde. The indicated NER repair factors were visualized by immunofluorescent double labeling using factor-specific antibodies. (B) Stable association between core TFIIH and CAK complex in HeLa cells. HeLa cells were either unirradiated or irradiated with 20 J/m2 UV and incubated in a fresh medium for 1 h. Whole cell extracts were made and IP was performed using the indicated antibodies. The immunoprecipitates were analyzed by Western blotting for XPB, p62 MAT1 and Cdk7 with specific antibodies. (C) Immunoslot-blot analysis of ChIP-recovered DNA. Unirradiated or UV-irradiated (20 J/m2) HeLa cells were cultured for 1 h before fixation with 1% formaldehyde. The soluble chromatin was made by sonication and, ChIP was performed with anti-XPB, anti-Cdk7 or anti-TFIID antibodies. The DNA from ChIP was recovered and isolated, and the DNA lesions were detected by immunoslot-blot analysis with anti-CPD antibody. Genomic DNA samples isolated from both unirradiated and UV-irradiated cells were used as controls for estimating the enrichment of UV DNA lesions in ChIP-recovered DNA.