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First published online 15 December 2004
doi: 10.1242/jcs.01603

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Localisation of human Y-family DNA polymerase : relationship to PCNA foci

Tomoo Ogi, Patricia Kannouche^* and Alan R. Lehmann

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RR, UK

View larger version (45K): [in a new window]   Fig. 1. Localisation of pol following DNA damage. (A) Foci formation frequencies of pol after DNA damaging (UV irradiation, BaP and -irradiation) or replication inhibitory (HU) treatments. MRC5 cells from our laboratory (GDSC, open bars) or from the laboratory of Bergoglio and colleagues (Tou, shaded bars) (Bergoglio et al., 2002) were transfected with eGFPpol constructed in our laboratory (GFPK, black), eGFP-C2-HsPOLK constructed in the laboratory of Bergoglio (GFPK-Tou, blue), or eGFPpol (GFPH, red) and incubated for 20 hours. Cells were then treated with the indicated doses of damaging agents and further incubated for the indicated times. The proportion of eGFPpol (or eGFPpol)-expressing cells in which the protein was localised in nuclear foci was determined. All experiments were carried out in triplicate and each data point is the mean of three independent scorings. Benzo[a]pyrene (BaP) and -ray experiments were carried out only with cells and plasmid from our laboratory. Error bars indicate standard error. (B,C) Typical images of cells expressing eGFPpol (B), or eGFPpol (C) 6 hours after 10 J/m2 UV irradiation. (D) Sub-nuclear pol protein localisation after UV irradiation. MRC5 cells were transfected with pEGFPpol, or pEGFPpol and incubated for 20 hours. Cells were then UV irradiated with 10 J/m2 and incubated for 6 hours. Cellular proteins were fractionated into detergent extractable (unbound, UB), salt extractable nuclear matrix binding fraction (NcB) and a fraction resistant to salt extraction (chromatin binding, ChrB) and analysed by immunoblotting.

View larger version (55K): [in a new window]   Fig. 2. Localisation of pol and PCNA after UV irradiation. MRC5 cells were transfected with pEGFPpol and incubated for 20 hours. Cells were UV irradiated with 10 J/m2 and incubated for 6 hours. Cells were then fixed and stained with anti-PCNA mouse monoclonal antibody and rhodamine-conjugated secondary antibody. Colocalisation of eGFPpol and PCNA foci was analysed. (A) eGFPpol-expressing cells were selected and sorted by the foci formation property of pol and PCNA. To check the colocalisation of foci, images of the cells that form both eGFPpol and PCNA foci were captured and then further analysed. Experiments were carried out in triplicate, and indicated numbers are the averages and the standard deviations of three independent experiments. More than 100 cells were analysed in each experiment. (B-E) Typical staining patterns of the auto-fluorescent signal of eGFPpol (green) and PCNA (red) in the same cell are shown. (B) Complete colocalisation of eGFPpol foci and PCNA foci was observed as shown by yellow staining. (C) Partial colocalisation of eGFPpol foci and PCNA foci. Most of the PCNA foci colocalised with eGFPpol foci, but significant numbers of eGFPpol foci were missing (white arrows). (D) eGFPpol foci were completely absent in cells with PCNA foci. (E) eGFPpol foci and PCNA foci were not colocalised at all.

View larger version (42K): [in a new window]   Fig. 3. The C-terminal region of pol protein is required for foci formation. MRC5 cells were transfected with plasmids expressing various eGFP-tagged pol deletion proteins and incubated for 20 hours. Cells were then UV irradiated with 10 J/m2 or 10 mM HU and further incubated for 6 hours. (A) Summary of foci formation and cellular localisation properties of C-terminal truncation mutants. (B) Summary of foci formation and cellular localisation properties of N-terminal truncation mutants. C, cytoplasmic; C2HC, C2HC type Zinc finger domain; N, nuclear; NLS, nuclear localisation signal like domain; PC, similar to PCNA binding domain consensus; *1, both nuclear and cytoplasmic localisation, but majority was nuclear; *2, both nuclear and cytoplasmic localisation. (CI) Typical images of UV-irradiated cells expressing eGFP-tagged pol deletion proteins.

View larger version (20K): [in a new window]   Fig. 4. pol foci formation is independent of pol. Pol-deficient XP30RO cells were transfected with pEGFPpol (GFPK, open bars), pEGFPpol (GFPH, filled bars). 20 hours later, cells were UV irradiated with 10 J/m2 and then incubated for a further 6 hours. The proportion of cells expressing eGFPpol or eGFPpol in which the protein was localised in nuclear foci was determined. All the experiments were carried out in triplicate and each data point is the mean of three independent scorings. Error bars indicate standard error.

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