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doi: 10.1242/10.1242/jcs.00162

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Localisation of human DNA polymerase to replication foci

Genetic Instability and Cancer, Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex, France

View larger version (31K): [in a new window]   Fig. 1. Localisation of eGFP-pol in the whole MRC5 cells (A) and colocalisation of eGFP-pol with PCNA in 20% MRC5 cells corresponding to the cell population in S phase (B). MRC5 cells were transfected with plasmid peGFP-C2 or peGFP-C2/pol. Cells were then fixed, stained with anti-PCNA mAb, incubated with TRITC-conjugated secondary antibody, and analysed by using confocal microscopy for the localisation of either eGFP or eGFP-pol fluorescent proteins or PCNA. The localisation of eGFP-pol (green) and PCNA (red) was observed in the same cell and the colocalisation is indicated by the yellow pattern (Merge). About 1000 fluorescent cells were analysed. DNA content was evaluated by analysing 20,000 collected events by flow cytometry (C).

View larger version (100K): [in a new window]   Fig. 2. Association of Pol with the replication machinery. Cells were transfected with peGFP-C2/pol and treated for 12 hours with 1.5 mM HU (A,B). HU was maintained (A) or chased by rinsing cells with fresh medium and then incubated for 8 hours (B). Cells were fixed and stained with anti-PCNA mAb followed by TRITC-conjugated secondary antibody. The localisation of eGFP-pol (green) and PCNA (red) was observed in the same cell and the colocalisation is indicated by the yellow pattern (Merge). About 1000 fluorescent cells were analysed.

View larger version (100K): [in a new window]   Fig. 3. Formation of eGFP-pol foci in UV or cisplatin-treated cells. Cells were transfected with peGFP-C2/pol and UV-irradiated 20 hours later at 10 J/m2 dose (A) or treated for 1 hour with 30 µM cisplatin (B). Cells were fixed and stained with anti-PCNA mAb followed by TRITC-conjugated secondary antibody. The localisation of eGFP-pol (green) and PCNA (red) was observed in the same cell and the colocalisation is indicated by the yellow pattern (Merge). About 1000 fluorescent cells were analysed.

View larger version (18K): [in a new window]   Fig. 4. Relocalization of eGFP-pol in UV-treated MRC5 and XP12RO cells. Twenty hours after DNA transfection, peGFP-C2/pol-containing cells were irradiated at 0 J/m2, 2 J/m2, 5 J/m2 or 10 J/m2 and incubated for 6 hours at 37°C, 5% CO2. About 1000 fluorescent cells were then analysed as already described.

View larger version (18K): [in a new window]   Fig. 5. Chromosomal mutagenesis in Pol-overexpressing cells. Pol overexpression was estimated by semi-quantitative RT-PCR (A). A 30-cycle amplification led to a saturating signal (ND, not detectable), whereas a 10- or a 20-cycle procedure allowed measurement of the signal overexpression ratio between RNA extracts from untreated and doxycyclin-treated pTet-Off/pTRE2-pol-8-4-containing cells. (B) Untransfected MRC5 cells or MRC5 cells containing either pTet-Off/pTRE2 (8-TRE2) or pTet-Off/pTRE2-pol (8-4) plasmids were grown in the presence or absence of doxycycline and then incubated with 6-thioguanine to select hprt mutants. Errors were calculated from three independent experiments.