Cell cycle-dependent resolution of DNA double-strand breaks

Abstract

DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are not repaired and maintain a sustained activation of the p53-pathway. Conversely, re-entry into cell cycle of damaged G0-arrested cells, occurs with a delayed clearance of DNA repair factors initially recruited to DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1-synchronized cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar in asynchronously proliferating, G0-, or G1-synchronized cells. Our study thereby demonstrates that repair and resolution of DSBs is strongly dependent on the cell-cycle state.

Keywords: AsiSI restriction enzyme; DSB repair; cell-cycle; site-specific DSBs.

Figure 1. 4OHT treatment triggers DSBs formation at AsiSI sites in MCF10A

A. MCF10A-AsiSI-ER cells were treated for 2 h with 4OHT or vehicle (Untr) and then released into fresh medium for 4 and 8 h (Recovery). Cells were fixed and processed for anti-HA immunofluorescence and DAPI staining. B. MCF10A-AsiSI-ER cells were treated for 2 h with 4OHT and stained with anti-γH2AX antibody. DAPI staining of nuclei is shown. C. MCF10A-AsiSI-ER cells were treated as above and ChIP experiments were performed using antibodies against γH2AX, NBS1 and XRCC4. Real-time qPCR was done on ChIP materials using primers listed in Supplementary Table 2. Amplicon far from any AsiSI site was analyzed as negative control. Data are from independent experiments with SD (n = 3).

Figure 2. AsiSI-induced DSBs trigger DDR activation followed by efficient wave of repair

A. Cell cycle distribution of asynchronously growing MCF10A-AsiSI-ER treated for 2h with 4OHT then released into fresh medium and collected as indicated. DNA content of propidium iodide stained cells was determined by flow cytofluorimetry. B. Total cell extracts from proliferating MCF10A-AsiSI before and at the indicated times after 4OHT removal were probed with anti-phospho-p53 and normalized for actinin. C. ChIP against γH2AX and NBS1 in MCF10A-AsiSI-ER treated for 2h with 4OHT then released into fresh medium, collected as indicated and analyzed by qPCR. Data are from independent experiments with SD (n = 3).

Figure 3. ChIP-seq analyses in proliferating and G₀-arrested MCF10A-AsiSI-ER cells after 4OHT treatment (2 h), using anti-γH2AX antibody

Panel A. show the profiles of γH2AX around a selected AsiSI site in both proliferating and G₀-arrested cells. B. Averaged γH2AX signals of proliferating and G₀ cells over a 10-kb windows and centered at the AsiSI site.

Figure 4. G₀-arrested MCF10-AsiSIER cells lack DNA repair proficiency

A. MCF10A-AsiSIER cells were arrested in G₀ phase through grow factors deprivation for 40h, treated with 4OHT for 2h then kept in medium without grow factors, and analyzed at the indicated times after 4OHT removal by immunofluorescence with anti-53BP1 and anti- γH2AX antibodies, respectively. B. Recruitment of γH2AX at AsiSI sites (Chr. 1 and 6) was determined by ChIP assays. C. Western blotting was performed using phospho-p53 antibodies and p21. D. PARP1 detection of both full-length and cleaved protein fragments; western blotting of G₀-arrested MCF10-AsiSI-ER treated with 4OHT or vehicle, collected at the indicated time points after 4OHT removal. E. DDR factors mRNAs expression analysis of G₀-arrested MCF10-AsiSI-ER through quantitative RT-PCR. Expression profiles were normalized against proliferating cells. F. Western blot of protein extracts of Growing, G₀ and G1/S MCF10-AsiSI-ER cells using the indicated antibodies. Actinin has been probed as loading control for different blots.

Figure 5. Cell-cycle re-entry induces a delayed resolution of DSBs

In panel A and B. cell cycle profiles and Ki67 levels detected by flow cytofluorimetry of G₀ MCF10-AsiSI-ER released into fresh medium and collected as indicated. C. Cell cycle distribution of G₀-arrested MCF10A-AsiSI-ER cells treated for 2h with 4OHT, then released into fresh medium and collected as indicated. DNA content of propridium iodide stained cells was determined by flow cytofluorimetry. D. Western blotting of MCF10A-AsiSI-ER cells treated as above. E. ChIP against γH2AX and NBS1 in MCF10A-AsiSI-ER treated for 2h with 4OHT, then released into fresh medium, collected as indicated and analyzed by qPCR. Data are from independent experiments with SD (n = 3).

Figure 6. DSBs induced at G1/S phase

Synchronized cells were exposed for 2 hr to 4OHT and then allowed to recovery for the indicted times. A. total cell extracts from G1/S phase MCF10A-AsiSI before and at the indicated times after 4OHT removal were probed with anti-phospho-p53 and actinin as loading control. Panel B. ChIP against γH2AX and NBS1 in MCF10-AsiSI-ER analyzed by qPCR. Data are from independent experiments with SD (n = 3).

Figure 7. ChIP analysis with γH2AX, NBS1 and XRCC4 antibodies in MFC10AsiSI-ER cells after a short pulse of 4OHT treatment (20′)

The values reported were calculated as percentage of input. Error bars indicate SD for three independent experiments.