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First published online 5 December 2006
doi: 10.1242/jcs.03312

Journal of Cell Science 120, 23-32 (2007)
Published by The Company of Biologists 2007
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UVA irradiation induces relocalisation of the DNA repair protein hOGG1 to nuclear speckles

Anna Campalans1,*, Rachel Amouroux1,*, Anne Bravard1, Bernd Epe2 and J. Pablo Radicella1,{ddagger}

1 Département de Radiobiologie et Radiopathologie, Commissariat à l'Energie Atomique, UMR 217 CNRS/CEA, 18 route du Panorama, 92265 Fontenay aux Roses, France
2 Institute of Pharmacy, University of Mainz, 55099 Mainz, Germany


Figure 1
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  		Fig. 1. UVA irradiation induces hOGG1 relocalisation to foci associated to the nuclear matrix. (A) A HeLa cell line expressing hOGG1-GFP was UVA (UVA) or mock (NI) irradiated and allowed to recover for 2 hours. Cells were then either directly fixed (-CSK) or pre-extracted with detergent-containing buffer (+CSK) before fixation. DNA was stained with DAPI. UVA-treated cells were further treated with DNase (absence of DAPI staining indicating the complete degradation of DNA) or RNase. (B) NI and UVA-irradiated cells were separated into fractions S1 (soluble proteins), P1 (proteins bound to DNA) and P2 (nuclear matrix proteins). Samples were analysed by western blot with anti-hOGG1 antibodies, detecting the fusion protein (upper panel) and, after overexposure, the endogenous hOGG1 (bottom panel). Middle panel shows the nuclear matrix protein Sm. (C) UVA-irradiated cells were allowed to recover for the indicated times. Soluble proteins were extracted with CSK buffer before fixation and DAPI staining. (D) Kinetics of hOGG1 accumulation in the P2 fraction after UVA irradiation. (E) UVA-irradiated cells were incubated at 37°C for 2 hours, and BrdU was added to the medium for the last 45 minutes. BrdU was visualised using an anti-BrdU antibody (red). Bars, 4 µm.

 

Figure 2
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  		Fig. 2. Subnuclear distribution of hOGG1-GFP in UVA-irradiated cells. (A) HeLa cells expressing hOGG1-GFP were UVA irradiated and incubated for 2 hours at 37°C. After fixation, DNA was stained with PI-RNase (red). (B) UVA-irradiated cells were immunostained with anti-B23 antibody (red). (C) UVA-irradiated and NI cells were immunostained with anti-SC35 (red), arrows indicate the absence of hOGG1-GFP in nuclear speckles in NI cells. The positions of the line scans shown in D are indicated in the merged images. (D) CCF analysis and line scans of hOGG1-GFP (green) and SC35 (red) signals. In UVA-irradiated cells a maximum overlap coefficient and a peak around {Delta}X=0 indicates a positively correlated, nonrandom colocalisation. In the line scan, the fluorescence intensity peaks for hOGG1-GFP and SC35 perfectly coincide, indicating the colocalisation of hOGG1-GFP with nuclear speckles. Conversely, in the NI cells, the CCF profile indicated an exclusion of hOGG1-GFP from nuclear speckles. Bars, 4 µm (A,B); 2 µm (C).

 

Figure 3
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  		Fig. 3. hOGG1 relocalisation to nuclear speckles is not dependent on the recognition of the lesion 8-oxoG. (A) Fpg- and T4-endoV-sensitive sites and single-strand breaks (ssb) were measured by alkaline elution in non-irradiated (NI) and UVA-irradiated cells. Bars represent the average of at least three determinations ± s.d. (B) HeLa cells were transiently transfected with the plasmids encoding hOGG1-GFP or the mutant proteins K249Q-GFP and F319A-GFP. Protein extracts were analysed by western blotting with an anti-hOGG1 antibody. (C) The same protein extracts were assayed for hOGG1 glycosylase activity. S and P indicate the substrate and the product, respectively. (D) Transiently transfected cells were UVA-irradiated and analysed under the confocal microscope. Both mutant proteins K249Q and F319A, as well as the wild-type hOGG1 (green), were able to re-localise into foci co-localising with SC35 (red). Bars, 4 µm.

 

Figure 4
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  		Fig. 4. Effect of transcription blockage on hOGG1 relocalisation to nuclear speckles. (A) Images corresponding to single optical sections from the middle of cells were thresholded and binarised and the percentage of round speckles calculated in NI and UVA-irradiated cells. Representative images are presented. (B) RNA synthesis was visualised by incorporation of BrU for 60 minutes in NI and UVA irradiated cells. BrU incorporation was detected with an antibody directed against BrdU (red). Arrows indicate nucleoli in the NI cells. (C) Cells were treated for 2 hours with the transcription inhibitors actinomycin-D (2 µg/ml) or {alpha}-amanitin (50 µg/ml) and immunostained with SC35 antibodies (red). hOGG1-GFP was excluded from speckles (see insets). (D) Actinomycin-D-treated cells were stained with PI-RNase (red). (E) hOGG1-GFP-expressing cells were UVC-irradiated and stained with the antibody against SC35 (red). Insets show the exclusion of hOGG1-GFP from nuclear speckles. Bar, 4 µm (B,C,E); 2 µm (D).

 

Figure 5
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  		Fig. 5. Oxidative stress induced by UVA triggers hOGG1-GFP recruitment to nuclear speckles. (A) Visualisation of ROS production in NI and UVA-irradiated cells by CH2-DCFDA green fluorescence. DNA was stained with DAPI. (B) Total, oxidised (GSSG) and reduced (GSH) glutathione levels were measured in NI and UVA-irradiated cells. Bars represent the average of two experiments ± s.d. (C) Addition of 2 mM of NAC during UVA irradiation and recovery periods prevents the formation of hOGG1 foci. DNA was stained with DAPI. Bars, 4 µm.

 

Figure 6
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  		Fig. 6. APE1 colocalises with nuclear speckles and hOGG1 in UVA-irradiated cells. (A) NI and UVA-irradiated HeLa cells expressing APE1-YFP, were fixed 2 hours after irradiation and immunostained with SC35 antibodies. Arrows indicate the colocalisation between APE1 and SC35 in UVA-irradiated cells. (B) HeLa cells were co-transfected with plasmids expressing APE1-YFP and hOGG1-DsRed. Two hours after UVA irradiation, cells were fixed and directly visualised. Arrows indicate colocalisation between hOGG1-DsRed and APE1-YFP. (C) Mock (NI) or UVA-irradiated cells expressing hOGG1-GFP were collected 2 hours after irradiation and in vivo crosslinked with 50 µg/ml DSP. Proteins associated with the nuclear matrix were extracted and equivalent amounts were analyzed by western blot with antibodies against hOGG1, APE1 and Lamin A/C. Bars, 2 µm.

 

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© The Company of Biologists Ltd 2007