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First published online 21 March 2006
doi: 10.1242/jcs.02868

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Spatiotemporal dynamics of p21^CDKN1A protein recruitment to DNA-damage sites and interaction with proliferating cell nuclear antigen

¹ Dipartimento di Medicina Sperimentale, sez. Patologia generale, Università di Pavia, 27100 Pavia, Italy
² Ludwig Maximilians University Munich, Department of Biology II, 82152 Planegg-Martinsried, Germany
³ Istituto di Genetica Molecolare-CNR, sez. Istochimica e Citometria, Dipartimento di Biologia Animale, Università di Pavia, 27100 Pavia, Italy
⁴ Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany

View larger version (59K): [in a new window]   Fig. 1. p21 is not completely degraded after UV-induced DNA damage. (A) Dose response analysis of p21 degradation after UV-induced DNA damage in LF1 human fibroblasts. Cells were lysed directly in loading buffer 6 hours after UV-C irradiation at the indicated doses. Samples were analysed by western blot for p21 protein levels versus actin as a loading control. (B) Time-course analysis of p21 degradation after UV irradiation at 2.5 or 10 J/m2. Samples were analysed for p21 and actin, as above.

View larger version (41K): [in a new window]   Fig. 2. Early recruitment of p21 to DNA repair sites. (A) LF1 human fibroblasts were irradiated with UV-C (10 J/m2) and 30 minutes later samples were extracted in situ and fixed for indirect immunofluorescence determination, or biotin-streptavidin amplification of chromatin-bound PCNA (green fluorescence) and p21 (red fluorescence), respectively. DNA (blue fluorescence) was stained with Hoechst 33258. (B) HeLa cells were cotransfected with p21-GFP and RFP-PCNA expression vectors, and 24 hours later exposed to UV-C radiation (10 J/m2). After 30 minutes, control (C) and irradiated (UV) cells were extracted in situ and fixed for detection and counting of cells showing only chromatin-bound RFP-PCNA (empty bars), p21-GFP (solid bars), or both (dashed bars). The percentages of cells in a representative experiment are shown. (C) Confocal sections of merged green and red fluorescence signals, of untreated control and UV-C-irradiated cells. (D) HeLa cells expressing p21-GFP and RFP-PCNA were exposed to local UV-C radiation (10 J/m2) through 3 µm pores. Confocal sections of green (p21-GFP) and red (RFP-PCNA) fluorescence signals are displayed, together with the merged image showing also the blue fluorescence (Hoechst) of DNA counterstaining. Bars, 10 µm (A,C); 5 µm (D).

View larger version (61K): [in a new window]   Fig. 3. Dynamics of p21 recruitment to DNA-repair sites in living cells. (A) C2C12 myoblasts expressing both p21-GFP and RFP-PCNA were exposed to 405 nm laser microirradiation and fluorescence signals were acquired after the indicated times. Maximum projections of confocal mid sections show the accumulation of p21-GFP and RFP-PCNA fluorescence signals at sites of DNA damage (arrows). (B) Short-term kinetic analysis of p21-GFP and RFP-PCNA fluorescence after 405 nm laser microirradiation. Signals were acquired every 2 seconds and confocal sections are shown of images taken at the indicated times. The arrows indicate the site of irradiation. (C) Plot of the relative fluorescence intensity of p21-GFP (green) and RFP-PCNA (red) at the irradiated spot. Fluorescence intensities acquired every 2 seconds at the irradiated region, were corrected for background, and for total nuclear loss of fluorescence over the time course, and normalised to the pre-irradiation value. Bars, 5 µm (A,B).

View larger version (69K): [in a new window]   Fig. 4. Induction of p21 expression does not inhibit recruitment of PCNA and DNA repair proteins. LF1 fibroblasts were treated for 16 hours with TSA to induce expression of p21, as described in the Materials and Methods. 30 minutes after exposure to UV-C radiation (10 J/m2), cells were directly lysed in loading buffer, for determination by western blot of total cellular content (total) of p21, PCNA, pol (p125 subunit) and Lig I. (A). Parallel samples were fractionated for western blot analysis of proteins in the chromatin-bound fraction (B). Actin was also determined as a loading control.

View larger version (80K): [in a new window]   Fig. 5. Colocalisation of p21-GFP with DNA repair proteins recruited to DNA-damage sites. HeLa cells expressing p21-GFP were locally irradiated with UV-C (10 J/m2) through 3 µm pores. After 30 minutes, cells were extracted in situ and fixed for determination of chromatin-bound p21-GFP and immunofluorescence staining of DNA repair proteins. (A) Confocal sections of p21-GFP (green) and pol (red) fluorescence signals are displayed together with the merged image also showing DNA counterstaining with Hoechst 33258 (blue). (B) confocal sections showing the single and merged images of p21-GFP (green), and XPG (red) at the irradiated sites. DNA was counterstained with Hoechst 33258 (blue). (C) Confocal sections showing the recruitment of p21-GFP (green) and CAF-1 (red), and DNA counterstaining (blue). Bars, 5 µm.

View larger version (73K): [in a new window]   Fig. 6. p21 does not displace pol from binding to PCNA after UV-C DNA damage. (A) Immunoprecipitation (Ip) was performed on detergent-soluble (S), or chromatin-bound fraction (Cb) obtained from LF1 fibroblasts irradiated or not with UV-C (10 J/m2), and harvested after 30 minutes. Samples were immunoprecipitated with anti-p21, or with anti-p125 (pol ) polyclonal antibodies, or with purified rabbit immunoglobulins (Ig) for specificity control. The immunoprecipitated material was analysed by western blot for the presence of PCNA, pol (p125 subunit), and p21. The position of each protein is shown together with Ig heavy chains (Ig h). Fractionated extracts (Input) were loaded (1/30 and 1/15 for S and Cb fractions, respectively) together with recombinant PCNA (PCNAr), and analysed by western blot for pol , PCNA, p21, and actin as a loading control. (B) Immunoprecipitation (IP) was performed on HeLa chromatin-bound extracts with anti-GFP antibody. Cell extracts were obtained from cells expressing pEGFP (GFP), or p21-GFP, irradiated or not with UV-C (10 J/m2) and harvested at times indicated below each panel. Western blot analysis of PCNA and pol , was performed on immunoprecipitated material. The position of each protein is shown together with Ig heavy chains (Ig h). Chromatin-bound extracts (Input) were loaded (1/15) on a parallel gel for western blot analysis of pol , PCNA, p21-GFP, and actin as a loading control.

View larger version (71K): [in a new window]   Fig. 7. p21 recruitment to DNA repair sites requires interaction with PCNA. HeLa cells were transfected with HA-tagged constructs for expression of wild-type p21 (p21HAwt) or a mutant form (p21HAmt) unable to bind PCNA (p21PCNA–). (A) 24 hours after transfection, cells were exposed to local UV irradiation (15 J/m2) through filters with 3 µm pores, extracted in situ 30 minutes later and fixed for immunofluorescence staining with anti-HA (green fluorescence) or anti-PCNA (red fluorescence) antibody. Confocal sections of each signal, together with the merged images, are shown. Bars, 5 µm. (B) Immunoprecipitation was performed with anti-HA antibody on detergent-soluble (soluble), and chromatin-bound (chrom.) fractions obtained from non-transfected (ntr) cells, and from cells expressing p21wt (wt), or p21PCNA– (mt) proteins. Immunoprecipitated material was analysed by western blot with anti-pol , anti-PCNA, and anti-HA antibodies. The position of each protein is shown together with Ig heavy (Ig h), and light (Ig l) chains. For detergent-soluble and chromatin-bound extracts, 1/30 and 1/15 respectively, were loaded (Input) on a parallel gel for western blot analysis of pol , PCNA, p21HA-tagged proteins, and actin as a loading control.

View larger version (100K): [in a new window]   Fig. 8. p21 recruitment to DNA-damage sites depends on DNA repair activity. LF1 and XP20PV (XPA) fibroblasts were exposed to local UV-C irradiation (15 J/m2) through filters with 3 µm pores, extracted in situ, and fixed at the indicated times for determination of chromatin-bound PCNA and p21. Samples were immunostained with anti-PCNA polyclonal, and anti-p21 monoclonal antibody, detected respectively with secondary antibody conjugated with Alexa Fluor 488 (green fluorescence) or Alexa Fluor 594 (red fluorescence); DNA was counterstained with Hoechst 33258 (blue fluorescence). Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 9. p21 does not inhibit UDS repair activity. (A) Untreated or TSA-treated LF1 fibroblasts were irradiated with UV-C (10 J/m2), and incubated with 100 µM BrdU for 3 hours. Cells were then fixed and immunostained with anti-BrdU antibody, a secondary biotinylated antibody followed by streptavidin-FITC. Fluorescence images of untreated (C), or TSA-treated cells (TSA) are shown together with samples exposed to UV radiation (UV), or exposed after TSA treatment (TSA+UV). (B) Normalised fluorescence intensity of BrdU immunofluorescence in G1-phase cells, measured by flow cytometry. Mean values ± s.d. (n=3) are reported. *P<0.05 compared with levels in the UV sample (Student's t-test). (C) HeLa cells expressing pEGFP, or p21-GFP, were UV-C irradiated (20 J/m2), incubated for 2 hours in [3H]thymidine and then fixed. Cells were immunostained with anti-GFP primary antibody and HRP-conjugated secondary antibody, and detected by immunoperoxidase staining (brown precipitate). UDS is denoted by the presence of nuclear autoradiographic granules. (D) Quantification of UDS grains in non-S-phase nuclei. Mean values of grain number (± s.d.) in duplicate samples, are reported. Bars, 10 µm (A,C).