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Nucleic Acids Res
2014 Jun 01;4210:6380-92. doi: 10.1093/nar/gku298.
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Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining.
Soni A
,
Siemann M
,
Grabos M
,
Murmann T
,
Pantelias GE
,
Iliakis G
.
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In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors.
Figure 1. (A) Representative images of IR-induced chromatid translocations (indicated by arrows) as scored in the experiments described here. (B) Formation of translocations in wt, Lig4−/−, Rad54−/− and Lig4−/−Rad54−/− MEFs scored at metaphase 1–4 h after exposure to 1 Gy IR. A protocol specifically allowing the analysis of events occurring in the G2 of the cell cycle was employed (see text). Statistical analysis of observed differences among mutants is provided in Supplementary Table S1. The number of translocations per cell in nonirradiated controls were 0.0085 ± 0.01, 0.02 ± 0.03, 0.01 ± 0.0 and 0.02 ± 0.0 in wt, Lig4−/−, Rad54−/− and Lig4−/−Rad54−/− MEFs, respectively. (C) Effect of PJ34, a specific Parp inhibitor, on translocation formation in wt, Lig4−/−, Rad54−/− and Lig4−/−Rad54−/− MEFs analyzed at metaphase 4 h after exposure to 1 Gy IR. Statistical analysis of the results obtained and the differences among mutants is given in Supplementary Table S2. (D) Phenotype validation of Parp-1−/− MEFs through H2O2-induced PAR staining. Note that in the H2O2-treated cells, PAR staining is abundantly present in the wt, but completely absent in the knockout mutant. (E) Translocations in wt and Parp-1−/− MEFs scored 4 h after exposure to 1 Gy IR. Cells were incubated in the presence or absence of the DNA-PKcs inhibitor, NU7441, to inhibit D-NHEJ and facilitate B-NHEJ. Statistical analysis of the results is provided in Supplementary Table S3. (F) Translocation formation in prematurely condensed chromosomes (PCC) of G2-phase V79 (wt) and irs1 (Xrcc2m) Chinese hamster cells 4 h after exposure to 1 or 5 Gy of IR. Results obtained with samples incubated in the presence of PJ34 are also shown for comparison. All data in this figure represent the mean ± SD calculated from three independent experiments.
Figure 2. (A) Formation of translocations in wt, Lig4−/−, Ku80−/−, DNA-PKcs−/−, Ku80−/−DNA-PKcs−/− MEFs, 1–4 h after exposure to 1 Gy IR. No translocations were detected in wt and Lig4−/− cells at 1 h post-IR. Statistical analysis and comparison of results obtained with the different mutants is provided in Supplementary Tables S4 and S5. (B) Translocation formation in wt, Lig4−/−, Ku80−/−, DNA-PKcs−/− and Ku80−/−DNA-PKcs−/− MEFs 4 h after exposure to 1 Gy IR. Results obtained with samples subject to incubation with PJ34 are also shown. Statistical analysis and comparison of the results obtained in the different sets is given in Supplementary Table S6. (C) HCT116 metaphase spreads subject to FISH for chromosomes 1 (green) and 2 (red) and showing chromosome translocations (indicated by white arrows) generated by exposure to 2 Gy IR. (D) Effect of Parp-1 inhibition on the frequency of chromosome translocations in HCT116 wt and Lig4−/− cells, 14 h after exposure to 2 Gy IR. Statistical analysis and comparison between the results obtained is given in Supplementary Table S7. Data represent the mean ± SD from two to three independent experiments.
Figure 3. (A) Effect of inhibition of DNA ligases on translocation formation in Lig4−/−Rad54−/− MEFs, as measured 4 h after exposure to 1 Gy IR. The inhibitors L82, L67 and a combination of PJ34 with L67 were administered 1 h prior to IR. The identity of the DNA ligases inhibited by each of these compounds is shown in the figure. Statistical analysis and intercomparison of the results obtained is provided in Supplementary Table S8. (B) Translocation formation in CHO (wt) and EM9 (Xrcc1 mutant) cells as measured at metaphase 4 h after exposure to 1 Gy IR. Results obtained with cultures exposed to PJ34 and NU7441 are also shown. (C) Effect of inhibition of DNA ligases 1 and 3 on translocations formation in CHO and EM9, as measured 4 h after exposure to 1 Gy IR. (D) Translocation formation in wt and Lig1−/− MEFs scored 4 h after exposure to 1 Gy IR. Cells were incubated in the presence or absence of the DNA-PKcs inhibitor, NU7441. Data represent the mean ± SD from two or three independent experiments.
Figure 4. (A) Metaphase spread of a wt MEF at 1 h after exposure to 1 Gy IR showing CBs (indicated by arrows). (B) Kinetics of CB repair in wt, Lig4−/−, Rad54−/− and Lig4−/−Rad54−/− MEFs, as measured 1–4 h after exposure to 1 Gy IR. The number of CBs per cell in 0 Gy controls were 0.14 ± 0.15, 0.063 ± 0.03, 0.04 ± 0.01 and 0.1 ± 0.05 in wt, Lig4−/−, Rad54−/− and Lig4−/−Rad54−/− MEFs, respectively. Statistical analysis and intercomparison of the results obtained is provided in Supplementary Table S9. (C) Effect of Parp inhibition on CB repair as measured in wt, Lig4−/−, Rad54−/− and Lig4−/−Rad54−/− MEFs, 1 or 5 h after exposure to 1 Gy IR. Statistical analysis and intercomparison of the results obtained is provided in Supplementary Table S10. Data represent the mean ± SD from three independent experiments.
Figure 5. (A) Representative two-parametric flow cytometry dot plots of Lig4−/− MEFs showing the activation of the G2-checkpoint by determining the MI, defined as the percent of cells at mitosis, through measurement of the fraction H3pS10 positive cells at 0, 1 and 5 h after exposure to 1 Gy IR. The x-axis shows the DNA signal measured by PI staining and the y-axis the intensity of the H3pS10 signal. Signal within the gates shown indicates that the corresponding cell entered mitosis. (B) Effect of Parp inhibition on G2-checkpoint response in wt MEFs after exposure to 1 Gy IR. (C) Effect of Parp inhibition on G2-checkpoint in Lig4−/− MEFs. Other details as in (B). The dotted and broken lines trace the results of wt cells and have been transferred from panel B. (D) As in (C) for Rad54−/− MEFs. (E) As in (C) for Lig4−/−Rad54−/− MEFs. Data represent the mean ± SD from three to four independent experiments. Where not visible, error bars are smaller than the symbols.
Figure 6. (A) G2-checkpoint response in CHO (wt) and EM9 (Xrcc1m) cells exposed to 1 Gy IR with or without NU7441 and/or L67 treatment. The percentage of mitotic cells was determined by counting about 1500–2000 Giemsa-stained cells per sample using bright field microscopy. (B) G2-checkpoint response in wt and Lig1−/− MEFs exposed to 1 Gy IR alone or in combination with NU7441. (C) G2 overlays for Rpa70 intensity showing chromatin bound Rpa in wt and Lig4−/−Rad54−/− MEFs 4 h after exposure to 10 Gy IR. The effect of Mre11 inhibition by Mirin on DSB end resection in Lig4−/−Rad54−/− MEFs is also shown. Fraction of G2 cells was assessed by their DNA content and gated using Kaluza 1.2 software to obtain G2 overlays depicting Rpa intensity. (D) Effect of Mirin on the translocation formation in Lig4−/−Rad54−/− MEFs. Results of a Mirin and PJ34 combination is also shown. Data represent the mean ± SD from two to three independent experiments.
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