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First published online 17 July 2007
doi: 10.1242/jcs.008367

Journal of Cell Science 120, 2706-2716 (2007)
Published by The Company of Biologists 2007
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Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC

Martijn S. Luijsterburg1,*, Joachim Goedhart2,*, Jill Moser3,*, Hanneke Kool3, Bart Geverts4, Adriaan B. Houtsmuller4, Leon H. F. Mullenders3, Wim Vermeulen5 and Roel van Driel1,{ddagger}

1 Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, Nuclear Organisation Group, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
2 Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, Section of Molecular Cytology and Center for Advanced Miscroscopy, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands
3 Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, PO Box 9600, 2300 RC Leiden, The Netherlands
4 Department of Pathology, Josephine Nefkens Institute, Erasmus MC, University Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands
5 Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands


Figure 1
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  		Fig. 1. Expression of DDB2-EYFP and EGFP-CUL4A. (A) Localization of DDB2 and CUL4A. The upper panel shows the distribution of DDB2-EYFP in an MRC5 cell, the middle image shows the distribution of EGFP-CUL4A in a HeLa cell and the lower images show the localization of endogenous DDB2 and CUL4A detected with specific antibodies in primary human cells (VH10). (B) Representation of the DNA constructs encoding fluorescently tagged DDB2 and CUL4A. (C) Western blot analysis of MRC5-SV40 cells expressing DDB2-EYFP. Whole cell extracts (10 µg) of non-UV-irradiated cells and UV-irradiated cells (2 and 4 hours after irradiation with 20 J/m2), were probed with antibodies against DDB2, DDB1 and CUL4A.

 

Figure 2
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  		Fig. 2. FRAP analysis of DDB2 mobility in non-damaged cells (NoDa). (A) Example of a DDB2-EYFP cell in which a strip (2 µm) spanning the nucleus was bleached. (B) Recovery plots of DDB2-EYFP (n=14) and XPG-EGFP (n=23), normalized between 0 and post-bleach intensity after full equilibration. (C) Normalized recovery plots of DDB2-EYFP at 37°C and 27°C (n=12), DDB2(R273H)-EYFP (n=13) at 37°C and EGFP-CUL4A (n=11) at 37°C. (D) Recovery of DDB2-EYFP normalized to pre-bleach intensity (red line). Monte Carlo simulation assuming a diffusion constant of 2.4 µm2/second (green line) and the residuals (blue lines at the bottom) that are a measure for the quality of the simulation. (E) Recovery of XPG-EGFP normalized to pre-beach intensity (red line), Monte Carlo simulation assuming a diffusion constant of 4.7 µm2/second (green line) and the residuals (blue lines).

 

Figure 3
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  		Fig. 3. Binding kinetics of DDB2 after local UV irradiation. (A) Example of a DDB2-EYFP cell that shows local accumulation after local UV-C irradiation with 100 J/m2 through 5 µm pores at 37°C. (B) Example of a HeLa cell expressing EGFP-CUL4A and (C) an XP-C cell expressing DDB2-EYFP. Both fusion proteins accumulate after local UV-C irradiation through 5 µm pores at 37°C (B,C). (D) Quantification of accumulation kinetics of DDB2-EYFP in MRC5 cells (brown line, n=9), EYFP-DDB2 in MRC5 cells (green line, n=8), DDB2-EYFP in XP-C cells (red line, n=3) and EGFP-CUL4A in HeLa cells (blue line, n=3). Curves were normalized to the plateau value. Time point t=0 is defined as the start of UV irradiation. (E) Quantification of assembly kinetics of DDB2-EYFP at 37°C (red, n=9) and 27°C (blue, n=8). The local accumulation of DDB2-EYFP was measured and plotted as a percentage of the total EYFP fluorescence in the cell nucleus. The error bars represent the standard deviation.

 

Figure 4
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  		Fig. 4. Nuclear distribution of DDB2 after global UV exposure. (A,B) Representative confocal images of DDB2-EYFP cells (C-E) Example of a DDB2-EYFP cell (C) that was transfected with cerulean-histone H2A (D); the merged images of the two signals is shown in E. (K) Line scan along the arrow in E. (F,G) Representative confocal images of DDB2-EYFP cells that were exposed to global UV-C irradiation (16 J/m2). (H-J) Globally UV-irradiated DDB2-EYFP expressing cell (H) that was transfected with cerulean-histone H2A (I); the merged images of the two signals is shown in J. (L) Line scan along the arrow in J. (M) Example of a cell expressing DDB2-EYFP, that was globally UV-C irradiated (16 J/m2) in metaphase and monitored until cell division was completed.

 

Figure 5
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  		Fig. 5. UV-induced degradation of DDB2. (A) Two DDB2-YFP cells were locally damaged by UV irradiation (nuclei indicated by the green dotted outline), while a third cell was not irradiated (indicated by the red dotted outline). The same three cells are shown shortly after (40 seconds) and 6 hours after UV irradiation. (B) Example of a DDB2-EYFP cell that has been globally UV irradiated (16 J/m2) and monitored for 5 hours. (C) Example of a globally UV irradiated (16 J/m2) DDB2-EYFP cell that had been treated with 50 µM MG-132. (D) Quantification of the total nuclear fluorescence in the absence of UV-C irradiation (green line, NoDa, n=8) and after global UV irradiation (GloDa) of MRC5 human cells with 4 (light-purple line, n=10), 8 (red line, n=12) and 16 J/m2 (light-blue line, n=10), and in XP-C (dark-blue line, n=15) and XP-A (dark-purple line, n=7) cells with 16 J/m2. All values were corrected for background fluorescence and the initial total nuclear fluorescence intensity was set to 100%. The error bars represent the standard error of the mean.

 

Figure 6
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  		Fig. 6. FRAP analysis of DDB2 mobility in UV-irradiated cells. (A) Example of a globally UV-irradiated DDB2-EYFP cell in which a narrow strip (2 µm) across the nucleus was bleached. (B) Recovery kinetics of DDB2-EYFP in undamaged cells (red line, NoDa, n=14), in locally damaged cells (green line, outside LoDa, n=14) and in globally UV-irradiated cells (4 J/m2; blue line, GloDa, n=15). The recovery plots are normalized to 0 and to the pre-bleach intensity. (C) The recovery plots of DDB2-EYFP in globally damaged cells 1 hour after (purple line, GloDa, n=16) and 4 hours after (blue line, GloDa, n=15) irradiation with 16 J/m2. The recovery plots of mutant R273H DDB2 (red line, n=12) and EYFP-histone H2A (green line, n=15) 1 hour after irradiation (16 J/m2) are also shown. The recovery plots are normalized to 0 and to the pre-bleach intensity. (D) Normalized recovery of DDB2-EYFP in cells irradiated with 4 J/m2 (blue line, GloDa). The recovery plot is normalized to post-bleach intensity and compared to DDB2-EYFP in undamaged cells (red line, NoDa). (E) UV-immobilized fraction of DDB2-EYFP in MRC5 cells 1 hour after global irradiation with 4, 8 and 16 J/m2 and 4 hours after 16 J/m2. The immobilized fractions depicted in E were determined by Monte Carlo simulations. The bound fractions obtained from the ten best simulations that fitted the experimental data were averaged. The error bars represent the standard deviation.

 

Figure 7
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  		Fig. 7. Dissociation kinetics of DDB2. (A) Example of a DDB2-EYFP cell after local UV irradiation with 100 J/m2. A strip of about one third of the cell nucleus opposite the local damage was continuously bleached and the fluorescence decrease in the local damage was monitored. (B) Quantification of the decrease in fluorescence signal in the damaged area of the nucleus. The half-time (t1/2) of a FLIP curve corresponds to the residence time of a protein molecule in the locally damaged area. FLIP was performed on DDB2-EYFP in MRC5 human fibroblasts at 37°C (purple line, n=10) and at 27°C (orange line, n=10), DDB2-EYFP in XP20MA human XP-C fibroblasts (blue line, n=11), XPF-EGFP in UV135 Chinese hamster ovary XP-G cells (green line, n=8) and XPC-EGFP in XP4PA human XP-C fibroblasts (red line, n=9). The error bars represent the standard error of the mean.

 




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