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. 2013 Nov 20:3:3277.
doi: 10.1038/srep03277.

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

Anna M Sokol  1 , Séverine Cruet-Hennequart, Philippe Pasero, Michael P Carty
Affiliations

"VSports最新版本" DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells

Anna M Sokol et al. Sci Rep. .

Abstract (V体育2025版)

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη VSports手机版. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy. .

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Conflict of interest statement

The authors declare no competing financial interests.

VSports最新版本 - Figures

Figure 1
Figure 1. Cell cycle progression and DNA replication analysis in carboplatin- and cisplatin-treated cells lacking or expressing DNA polymerase η.
Cells expressing (TR30-2) or lacking (XP30RO) polη were treated for 24 h with the indicated doses of cisplatin and carboplatin. Cells were incubated with 10 μM BrdU before fixing and staining with propidium iodide (PI) and anti-BrdU-FITC antibody as described in Materials and Methods. (a) Representative flow cytometry (PI- and BrdU-stained) dot plots are presented. Populations of cells with S-phase DNA content were divided in two groups termed normal replication and inhibited replication, based on BrdU staining. Asterisks indicate statistically significant differences in the percentage of cells exhibiting replication inhibition, determined using the two-way ANOVA test. (b) The mean percentage of cells in the indicated groups +/− SEM values, derived from four independent experiments, is presented in Table format.
Figure 2
Figure 2. Analysis of DNA replication by DNA combing.
(a) A schematic outline of the DNA combing protocol is shown. (b) DNA fibres were visualized using specific primary antibodies and fluorescently-labelled secondary antibodies. A representative fibre is shown. Single-stranded DNA is shown in blue, IdU-labelled DNA in red, and CldU-labelled DNA in green. (c) Representative, individual DNA fibres for each experimental condition are presented. (d) The frequency distribution of fibres of particular lengths is presented as histograms plots. Data represent the population of fibres obtained by measuring at least 150 individual DNA fibres for selected experimental conditions, in two independent experiments. The red line indicates a Gaussian curve fitted to the data. (e) Data are presented as inbox-and-whiskers graphs. Each box represents the 25th and 75th percentile (the lower and upper quartiles, respectively). The median (50th percentile) is marked as the line near the middle of the box. Data not included between the whiskers are plotted as outliers (dots). Data was analyzed by the use of the Mann-Whitney test. Asterisks indicate statistically significant differences.
Figure 3
Figure 3. Carboplatin- and cisplatin-induced DNA damage responses and apoptosis induction in cells lacking and expressing DNA polymerase η.
(a) Polη-deficient XP30RO cells and polη-expressing TR30-2 cells were treated with carboplatin or cisplatin and harvested 24 h post-treatment. Polη, DNA damage response proteins and apoptosis-related proteins were analysed by western blotting using specific antibodies, as described in Materials and Methods. For direct comparison, samples from both cell lines were run on the same gel. As a control for apoptosis induction, proteins, obtained from floating cells following treatment of cells with 5 μg/ml cisplatin for 24 h, were loaded in the lanes marked A. (b) Polη-deficient XP30RO cells were treated for 24 h with 8.5 μM cisplatin. Immunofluorescence microscopy was carried out using phospho-specific primary antibodies against γH2AX and phospho-Ser4/Ser8 RPA2, and Alexa 594- and Alexa 488-labeled secondary antibodies. DNA was counterstained with DAPI. Scale bar corresponds to 25μm.
Figure 4
Figure 4. Analysis of DNA replication by quantitative immunofluorescence.
Cells expressing (TR30-2) or lacking (XP30RO) polη were grown on glass coverslips and treated with the indicated doses of cisplatin or carboplatin. 75 minutes before fixing, cells were incubated with 10 μM EdU. Click chemistry was used to visualise EdU incorporation, and immunofluorescence staining was carried out using anti-phospho-Ser4/Ser8 RPA2 primary antibody and Alexa 594-coupled secondary antibody. DNA was counterstained with DAPI. (a) Representative images from three independent experiments are presented. The scale bar corresponds to 25 μm. Arrows show representative cells which exhibit decreased EdU incorporation and increased phospho-Ser4/Ser8 RPA2 staining. (b) The EdU fluorescence signal was quantified as described in Materials and Methods. Relative EdU fluorescence values were analysed using the Mann-Whitney test; significant values are marked with asterisks. In the inbox-and-whiskers graphs, the top and bottom of each box represents the 25th and 75th percentile (the lower and upper quartiles, respectively). The median (50th percentile) is marked by the line near the middle of the box. (c) Cells positive for phospho-Ser4/Ser8 RPA2 were counted in the EdU-positive cell population. Data represent the mean from three independent experiments +/− SEM. Statistically significant differences are marked with asterisks, as determined using a two-way ANOVA test. (d) EdU staining in phospho-Ser4/Ser8 RPA2-positive cells was analysed under conditions where RPA2 phosphorylation on Ser4/Ser8 is enriched. The intensity of the EdU signal in phospho-Ser4/Ser8 RPA2-positive cells was further analysed, and categorised into values above and below the threshold value. For each condition, the threshold value was defined as the mean relative EdU intensity value from the entire EdU-positive cell population, minus twice the SEM value. Data represent the mean percentage of cells in defined categories obtained in three independent experiments +/− SEM. Statistically significant differences for both the mean EdU intensity in each category as well as mean percentage of each category are marked with asterisks.
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