Influence of XPB helicase on recruitment and redistribution of nucleotide excision repair proteins at sites of UV-induced DNA damage

Abstract

The XPB DNA helicase, a subunit of the basal transcription factor TFIIH, is also involved in nucleotide excision repair (NER). We examined recruitment of NER proteins in XP-B cells from patients with mild or severe xeroderma pigmentosum (XP) having different XPB mutations using local UV-irradiation through filters with 5 microm pores combined with fluorescent antibody labeling. XPC was rapidly recruited to UV damage sites containing DNA photoproducts (cyclobutane pyrimidine dimers, CPD) in all the XP-B and normal cells, thus reflecting its role in damage recognition prior to the function of XPB. Cells from the mild XP-B patients, with a missense mutation, showed delayed recruitment of all NER proteins except XPC to UV damage sites, demonstrating that this mutation impaired localization of these proteins. Surprisingly, in cells from severely affected patients, with a C-terminal XPB mutation, XPG and XPA proteins were normally recruited to UV damage sites demonstrating that this mutation permits recruitment of XPG and XPA. In marked contrast, in all the XP-B cells recruitment of XPF was absent immediately after UV and was delayed by 0.5 and 3 h in cells from the mild and severely affected XP patients, respectively. Redistribution of NER proteins was nearly complete in normal cells by 3 h but by 24 h redistribution was only partially present in cells from mild patients and virtually absent in cells from the severely affected patients. Ineffectual repair of UV-induced photoproducts resulting from delayed recruitment and impaired redistribution of NER proteins may contribute to the markedly increased frequency of skin cancer in XP patients.

Fig. 1

Delayed repair of 6–4 photoproducts (6–4PP) and cyclobutane pyrimidine dimers (CPD) in cells from XP-B patients. XP and normal cells were irradiated with 10 J/m² UV and incubated for the indicated times. The levels of 6–4PP (A) and CPD (B) were measured by ELISA using either anti-6–4PP or anti-CPD antibodies. Each point represents the mean from four independent experiments. The points are displaced slightly to permit visualization of the S.D. error bars.

Fig. 2

Recruitment and redistribution of NER proteins to sites of UV damage. Normal (0.8 µm beads) and XP-B cells (2.0 µm beads) on same slide were irradiated with 100 J/m² UV through 5 µm pore size filters and immediately fixed (<0.1 h) or subsequently cultured for various time points before fixation. Immunofluorescent double labeling was performed using antibodies against CPD, XPC, XPG, XPA, XPD or XPF. The arrows on the confocal images indicate sites of localized damage (red arrows, normal cells; yellow arrows, XP-B cells). Symbols + or − indicate localization or non-localization of NER protein in normal cells/XP-B cells. (A) Delayed recruitment of XPG, XPA, XPD and XPF proteins to damage sites in XP33BR. (B) Normal recruitment of XPC, XPG and XPA proteins to damage sites in XP131MA cells. The recruitment of XPD was delayed and no XPF recruitment was seen 0.5h after UV-irradiation. NER proteins in all XP-B cells remained visible at damage sites even 24h after local UV-irradiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Quantification of recruitment and redistribution of NER proteins to sites of UV damage at various times post-UV-irradiation. Images of slides as in Fig. 2 were analyzed. At least 100 nuclei were scored for each time point. Bars indicate mean ± S.E. of the percent positive cells for the indicated antibody stain (CPD, XPC, XPG, XPA, XPD or XPF, respectively).

Fig. 4

Enhanced recruitment of XPB and XPF in XP183MA cells transiently transfected with wild-type XPB-cDNA. Normal (AG13145) and XP183MA cells from a severe XPB patient were either not over-expressed or over-expressed with wild-type XPB cDNA. (A) Western blotting of XPB protein in normal and XP183MA cells transiently transfected with wild-type XPB-cDNA or empty vector. Blots were probed with antibody to the C-terminal end of XPB or to β-actin. (B) Immediately (<0.1 h) after local UV-irradiation recruitment of XPB or XPF was detected in normal cells (red arrows) but not in XP183MA cells treated with the empty vector. (C) In contrast, normal recruitment of XPB and XPF was detected in XP183MA cells transiently over-expressing wild-type XPB-cDNA (yellow arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 5

Effect of XPG and XPA mutations on recruitment of NER proteins. Normal (0.8 µm beads) and NER deficient XP-G, XP-A or XP-B cells (2.0 µm beads) were treated as in Fig. 2 and fixed immediately (<0.1 h) after UV exposure. Normal cells showed rapid nuclear recruitment of XPC, XPG, XPA, XPB and XPF proteins to the UV damage sites (red arrows). XP-G (XP96TA) cells showed normal recruitment of XPC, XPA, XPB and XPF (yellow arrows) but no staining for XPG protein. In XP-A (XP315BE) cells, normal recruitment of XPC, XPB and XPG was detected (yellow arrows). There was no staining for XPA protein and no recruitment of XPF to the sites of UV damage. In the XP-B cells (XP131MA) from the severe patient, normal recruitment of XPC, XPG and XPA was detected (yellow arrows) (see also Fig. 2B). There was no staining for XPB and no recruitment of XPF protein was detected. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 6

Schematic diagram of early stages (<0.1 h) of global genome nucleotide excision repair in cells from normal, mild XP-B and severe XP-B patients. DNA damage recognition is accomplished by binding of DDB2 (XPE) – DDB1 proteins (purple ovals) to the 6–4 PP or the CPD (red circle) along with the XPC-HR23B-centrin2 complex (blue oval). In the normal cells (left column) this is followed by unwinding of double stranded DNA which is associated with binding of XPG (green circle), XPA (orange oval), and the TFIIH complex (light green) including XPB (dark pink oval) and XPD (pink oval). Dual incisions excising the DNA damage in an approximately 30 nucleotide segment are carried out by the ERCC1-XPF 5′ endonuclease (gray ovals with scissors) and the XPG 3′ endonuclease (green circle with scissors). In the cells from the mild XP-B patients with a F99S mutation (middle column) there is normal recruitment of XPC to the site of the DNA damage but there is no recruitment of XPG, XPA, XPD or XPF. In contrast, the cells from the severe XP-B patients (right column) with a C-terminal mutation show normal recruitment of XPC, XPG and XPA but no recruitment of XPD or XPF to the site of the DNA damage. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)