Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair

Abstract

Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER.

Fig. 1

XP-B and XP-B/CS fibroblasts are defective in accumulation of CAK component MAT1 at DNA damage sites in vivo. Normal human fibroblasts (NHF) (A), XP-B fibroblasts XP33BR (B), XP-B/CS fibroblasts XPCS1BA (C) and XP183MA (D) were grown on coverslips, locally irradiated with 100 J/m² UV through a 5 μm-isopore polycarbonate filter, fixed immediately (0 h) or cultured for 0.5, 3, or 24 h before fixing in 2% paraformaldehyde. The TFIIH component p62 and CAK component MAT1 were visualized by immunofluorescence double labeling using specific antibodies. Nuclei shown in blue are from counterstaining with DAPI. Arrows indicate immunofluorescent foci due to accumulation of THIIH proteins at localized damage sites.

Fig. 1

XP-B and XP-B/CS fibroblasts are defective in accumulation of CAK component MAT1 at DNA damage sites in vivo. Normal human fibroblasts (NHF) (A), XP-B fibroblasts XP33BR (B), XP-B/CS fibroblasts XPCS1BA (C) and XP183MA (D) were grown on coverslips, locally irradiated with 100 J/m² UV through a 5 μm-isopore polycarbonate filter, fixed immediately (0 h) or cultured for 0.5, 3, or 24 h before fixing in 2% paraformaldehyde. The TFIIH component p62 and CAK component MAT1 were visualized by immunofluorescence double labeling using specific antibodies. Nuclei shown in blue are from counterstaining with DAPI. Arrows indicate immunofluorescent foci due to accumulation of THIIH proteins at localized damage sites.

Fig. 2

Physical association of MAT1 with core TFIIH in XP-B and XP-B/CS fibroblasts. (A) Western blotting of XPB, XPD and MAT1 proteins in NHF, XP-B and XP-B/CS fibroblasts. Protein extracts were made in SDS lysis buffer from indicated normal and XP fibroblasts. The proteins were quantitated and resolved by polyacrylamide gel. The proteins transferred on blots were probed with antibodies to XPB, XPD, MAT1 or β-actin. (B and C) Whole cell extracts were made in RIPA buffer from NHF, XP-B and XP-B/CS fibroblasts. Immunoprecipitation was performed by using control (Ctrl) or MAT1 (B) or XPD (C) antibodies. The immunoprecipitates were analyzed by Western blotting with specific XPB, p62 or XPD antibodies.

Fig. 3

XP-D fibroblasts accumulate various pre-incision NER factors, including MAT1, at DNA damage sites in vivo. XP-D fibroblasts XP1BR (A) and XP17BE (B) were grown on coverslips, locally UV-irradiated and cultured for 1 h. The cells were then fixed with 2% paraformaldehyde and decorated by immunofluorescence double labeling with indicated specific antibodies. Nuclei were counterstained with DAPI. (C) Western blotting of XPB, XPD and MAT1 proteins in NHF and XP-D fibroblasts. Protein extracts were made in SDS lysis buffer, quantitated and resolved by polyacrylamide gel. The blots were probed with antibodies to XPB, XPD, MAT1and β-actin. (D) Physical association of MAT1 with core TFIIH in NHF and XP-D fibroblasts. Whole cell extracts were made in RIPA buffer from NHF and XP-D fibroblasts. Immunoprecipitation was performed by using control (Ctrl) or MAT1 antibodies and the immunoprecipitates were Western blotting analyzed for presence of XPB.

Fig. 3

XP-D fibroblasts accumulate various pre-incision NER factors, including MAT1, at DNA damage sites in vivo. XP-D fibroblasts XP1BR (A) and XP17BE (B) were grown on coverslips, locally UV-irradiated and cultured for 1 h. The cells were then fixed with 2% paraformaldehyde and decorated by immunofluorescence double labeling with indicated specific antibodies. Nuclei were counterstained with DAPI. (C) Western blotting of XPB, XPD and MAT1 proteins in NHF and XP-D fibroblasts. Protein extracts were made in SDS lysis buffer, quantitated and resolved by polyacrylamide gel. The blots were probed with antibodies to XPB, XPD, MAT1and β-actin. (D) Physical association of MAT1 with core TFIIH in NHF and XP-D fibroblasts. Whole cell extracts were made in RIPA buffer from NHF and XP-D fibroblasts. Immunoprecipitation was performed by using control (Ctrl) or MAT1 antibodies and the immunoprecipitates were Western blotting analyzed for presence of XPB.

Fig. 4

XPG recruitment at DNA damage sites is impaired in XP-B/CS fibroblasts from patients with mild symptoms while persists in fibroblasts from patients with severe symptoms. NHF (A), XP-B/CS fibroblast XPCS2BA (B), XP183MA (C) were grown on coverslips and irradiated with 100 J/m² UV through a 5 μm-isopore polycarbonate filter. The cells were fixed immediately (0 h) or cultured for indicated repair period and then fixed in 2% paraformaldehyde. The XPG and XPB recruitment at DNA damage sites were examined by immunofluorescence double labeling as described for Fig. 1 to 3. The presence of p62, XPD and their co-localization at DNA damage sites were monitored in XP183MA fibroblasts (D).

Fig. 4

XPG recruitment at DNA damage sites is impaired in XP-B/CS fibroblasts from patients with mild symptoms while persists in fibroblasts from patients with severe symptoms. NHF (A), XP-B/CS fibroblast XPCS2BA (B), XP183MA (C) were grown on coverslips and irradiated with 100 J/m² UV through a 5 μm-isopore polycarbonate filter. The cells were fixed immediately (0 h) or cultured for indicated repair period and then fixed in 2% paraformaldehyde. The XPG and XPB recruitment at DNA damage sites were examined by immunofluorescence double labeling as described for Fig. 1 to 3. The presence of p62, XPD and their co-localization at DNA damage sites were monitored in XP183MA fibroblasts (D).

Fig. 5

UV-induced RNAPII Ser5-phosphorylation and degradation in XP-B/CS fibroblasts. The exponentially growing NHF (A), XPCS1BA, XPCS2BA (B), XP181MA and XP183MA (C) fibroblasts were UV-irradiated at a dose of 20 J/m² and maintained for indicated repair period in fresh medium. The protein extracts were then made in SDS lysis buffer, and the proteins were quantitated and examined by Western blotting using specific anti-XPB and anti-phospho-RNAPII or anti-β-actin antibodies. (D) Representative Western blot images were quantitated using ImageJ software and arbitrary amount of RNAPII were estimated relative to protein levels found without UV irradiation.