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First published online 5 August 2008
doi: 10.1242/jcs.031708

Journal of Cell Science 121, 2850-2859 (2008)
Published by The Company of Biologists 2008
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Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC

Deborah Hoogstraten1, Steven Bergink1, Jessica M. Y. Ng1,*, Vincent H. M. Verbiest1, Martijn S. Luijsterburg3, Bart Geverts2, Anja Raams1, Christoffel Dinant1,2, Jan H. J. Hoeijmakers1, Wim Vermeulen1,* and Adriaan B. Houtsmuller2,*

1 Department of Cell Biology and Genetics, Erasmus MC Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands
2 Department of Pathology (Josephine Nefkens Institute), Erasmus MC Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands
3 Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands


Figure 1
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  		Fig. 1. Characterization of the nuclear distribution of GFP-tagged XPC, XPA and TFIIH in living cells. (A) Immunoblot of whole cell extracts of MRC5 (lane 1), XP4PA SV (lane 2) and population of XP4PA SV cells stably expressing XPC-GFP (lane 3) probed with anti-XPC polyclonal antibodies. The asterisk shows a background band that can serve as a loading control. (B) UV survival of MRC5, XP4PA SV and XP4PA SV cells stably expressing XPC-GFP. The log of the percentage of survival is plotted against the dose of UV-C (J/m2). (C) Confocal image of a cell stably expressing XPC-GFP (left panel) and transiently expressing H2B-YFP (middle). The merged image is shown in the right panel. (D) A cell stably expressing XPB-GFP (left panel) and transiently expressing H2B-YFP (middle). (E) A cell stably expressing GFP-XPA (left panel) and transiently expressing H2B-YFP (middle). (F) Mitotic cell expressing XPC-GFP. Left panel, GFP fluorescence; middle, Hoechst 33342 staining of DNA; right, merged image.

 

Figure 2
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  		Fig. 2. Dynamics of XPC-GFP, TFIIH-GFP and GFP-XPA. (A) FRAP analysis of untreated GFP-XPA (blue line), XPB-GFP (red line) and XPC-GFP (green line) expressing cells. Error bars represent two times the s.e.m. based on a single experiment, n=24. Note that these experiments were repeated at least three times and consistently showed similar mobility differences. (B) Simultaneous FLIP-FRAP analysis on cells expressing GFP-XPA (blue line), XPB-GFP (red line) and XPC-GFP (green line) at 37°C. (C) Simultaneous FLIP-FRAP analysis on cells expressing GFP-XPA (blue line), XPB-GFP (red line) and XPC-GFP (green line) at 27°C. The curves (dashed) obtained at 37°C are also shown. (D) Simultaneous FLIP-FRAP analysis on cells expressing XPB-GFP (red lines) and XPC-GFP (green lines) in the presence and absence of DRB, an RNAP2 transcription inhibitor. (E) Simultaneous FLIP-FRAP analysis on GFP-XPA (blue lines) and XPC-GFP (green lines) expressing cells in the presence and absence of sodium azide.

 

Figure 3
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  		Fig. 3. Effect of DNA-structure-altering agents on XPC-GFP mobility. (A) Monitoring of the uptake of Hoechst 33342 by cells expressing XPC-GFP at different time points. Left panel, Hoechst 33342 staining of DNA; Middle, GFP-fluorescence; right, merged image. (B) Confocal images of a cell expressing XPC-GFP (upper panel), XPB-GFP (middle panel) or GFP-XPA (lower panel) stained with Hoechst 33342 for 30-60 minutes prior to fixation. Left panel, Hoechst staining of DNA; middle, GFP-fluorescence; right, merged image. (C) Strip-FRAP analysis of untreated (light green line) and Hoechst-33342-treated (dark green line) XPC-GFP-expressing cells. (D) Strip-FRAP analysis of untreated (light line) and Hoechst-33342-treated (dark line) cells expressing XPB-GFP (red lines) and GFP-XPA (blue lines). (E) Strip-FRAP analysis of untreated (light line) and actinomycin-D-treated (dark line) cells expressing XPB-GFP (red lines) and GFP-XPC (green lines). (F) Strip-FRAP analysis of untreated (green line), UV-A (light blue line) and UV-C-treated (dark blue line) XPC-GFP-expressing cells.

 

Figure 4
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  		Fig. 4. FRAP analysis of UV-C-treated cells expressing XPC-GFP. (A) FRAP analysis of untreated (green line) and UV-irradiated cells (blue lines) at different UV doses. (B) UV-dose-dependent and time-dependent immobilization of XPC-GFP. Percentage of UV-induced immobilization is plotted against time for the different UV-doses, non-damage-induced immobilization was set at zero. (C) Scheme of the FRAP-FLIP procedure on locally damaged areas. A small strip covering half of the local damage and spanning the entire nucleus is bleached at relatively low laser intensity for a period of 2 seconds. Subsequently fluorescence is monitored at regular time intervals in the bleached (FRAP) and non-bleached (FLIP) half of the local damage. (D) Confocal images of a locally irradiated cell expressing XPC-GFP (5 µm pore filter). Left panel, before bleaching; middle panel, directly after bleaching and right panel, 90 seconds after bleaching. (E) The relative fluorescence of the FRAP and FLIP area is shown over time. The log of fluorescence redistribution difference between FLIP and FRAP areas are plotted against time (dotted line). (F) Simultaneous FRAP/FLIP analysis of local damage at 37°C and 27°C. (G) Assembly kinetics of XPC-GFP to locally damaged areas at 37°C and 27°C. The curves are normalized to the bound fraction in the locally damaged area.

 

Figure 5
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  		Fig. 5. Shuttling of XPC-GFP between nucleus and cytoplasm. (A) Schematic representation of the XPC polypeptide, showing the different domains. (B) The three potential NES sequences present in XPC. (C) One nucleus of the polykaryon is bleached and subsequently the fluorescence in the bleached nucleus is followed over time. (D) The relative fluorescence of XPC-GFP in the bleached nucleus over time (light green line). For the control experiment, both nuclei are bleached (dark green line). (E) The relative fluorescence of XPC-GFP (green line), XPB-GFP (red line) and GFP-XPA (blue line) in the bleached nucleus over time. (F) The relative fluorescence of XPC-GFP in the bleached nucleus in untreated (green line) cells and in cells at different time points after UV irradiation of the entire cell population (blue lines).

 

Figure 6
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  		Fig. 6. Proposed model for XPC-GFP behavior in living cells. In nonirradiated cells, XPC constantly shuttles between nucleus and cytoplasm. In the nucleus, it interacts very transiently with DNA. In UV-irradiated cells, the shuttling is stopped, increasing the concentration in the nucleus. In addition, XPC continues probing DNA. Once damage is encountered, the binding time increases considerably, probably because of stabilization of the DNA-protein complex by binding of subsequent NER factors such as TFIIH and XPA.

 

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© The Company of Biologists Ltd 2008