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. 2022 Oct 20;82(20):3932-3942.e6.
doi: 10.1016/j.molcel.2022.08.028. Epub 2022 Sep 20.

DNA-PKcs promotes fork reversal and chemoresistance

Diego Dibitetto  1 , Shannon Marshall  2 , Andrea Sanchi  3 , Martin Liptay  4 , Jumana Badar  2 , Massimo Lopes  3 , Sven Rottenberg  5 , Marcus B Smolka  6
Affiliations

DNA-PKcs promotes fork reversal and chemoresistance

Diego Dibitetto et al. Mol Cell. .

Abstract

The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.

Keywords: BRCA1; BRCA2; DNA replication; DNA-PKcs; NHEJ; PARP inhibitor; chemoresistance; fork reversal.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. The DNA-PKcs-KU complex is broadly required for replication stress-induced fork slowing and reversal.
a) DNA-PKcs SIRF assay in RPE1 cells treated according to the indicated scheme. Dot plot shows the number of foci and the median from 180 cells in 3 biological replicates. **** p<0.001 One-way Anova test. Scale bar 15 μm. b) KU80 SIRF assay in RPE1 cells treated according to the indicated scheme. Dot plot shows the number of foci and the median from 180 cells in 3 biological replicates. **** p<0.001 One-way Anova test. Scale bar 15 μm. c) DNA fiber analysis in U-2OS cells treated according to the depicted scheme. At least 350 individual fibers for each condition were scored. Dot plot and median of total tract length are shown (n=3). **** p<0.001 One-way Anova test. d) DNA fiber analysis in RPE1 cells treated according to the depicted scheme. Dot plot shows the IdU/CldU ratio from 200 individual fibers scored from 2 biological replicates. **** p<0.001 One-way Anova test. e) DNA fiber analysis in U-2OS cells transfected with the indicated siRNA and treated according to the indicated scheme. 300 individual fibers for each condition were scored. Dot plot and median of total tract length are shown (n=3). **** p<0.001 One-way Anova test. f) DNA fiber analysis of U-2OS cells treated for 5 days with cATRi (AZD6738 0.4μM) followed by 6 hours with 5μM NU7441. 300 individual fibers for each condition were scored. Dot plot and median of total tract length are shown (n=3). **** p<0.001 One-way Anova test. g) EM analysis of reversed forks following the same conditions as in (f). Electron micrograph represents a reversed replication fork. P, parental strand; D, daughter strand; R, regressed arm. The graph-bar shows the mean of reversed forks frequency from two independent EM experiments. P-values are calculated with unpaired two-tailed Student’s t-test. h) DNA fiber analysis in RPE1 cells treated according to the indicated scheme. Dot plot shows the individual IdU/CldU ratios and the median from 200 individual fibers scored from 2 biological replicates. **** p<0.001 One-way Anova test.
Figure 2.
Figure 2.. DNA-PKcs promotes fork degradation in BRCA1-deficient cells.
a) Schematic of a DNA fiber assay to monitor fork degradation in BRCA-deficient cells. b) DNA fiber analysis in U-2OS cells transfected with the indicated siRNA according to the depicted scheme. Dot plot shows the IdU/CldU ratio and the individual IdU tract length from 300 individual fibers and the median from 3 biological replicates. **** p<0.001 unpaired One-way Anova test. c) DNA fiber assay performed as in (b). Dot plot shows IdU/CldU ratio from 300 individual fibers from 3 biological replicates. **** p<0.001 unpaired One-way Anova test. d) RAD51 foci analysis in U-2OS cells transfected with the indicated siRNA and treated as indicated. Bar graph shows the percentage of γH2AX+ and RAD51+ cells from 3 independent biological replicates. P-values were calculated with the One-way Anova test. Scale bar 10μm. e) Resection assay. ssDNA was measured at Cas9-induced breaks in U-2OS TetON-eGFP-Cas9 cells transfected with the indicated siRNA. Bar graph shows the percentage of ssDNA at 364bp from Cas9 site from 2 independent biological replicates. P-values are calculated with unpaired two-tailed Student’s t-test. f) DNA fiber analysis in U-2OS cells done as in (b). Dot plot shows the IdU/CldU ratio and the individual IdU tract length from 300 individual fibers and the median from 3 biological replicates. **** p<0.001 unpaired One-way Anova test. g) DNA fiber analysis in U-2OS cells done following the depicted scheme. Dot plot shows IdU/CldU ratio from 300 individual fibers and the median from 3 biological replicates. **** p<0.001 unpaired One-way Anova test.
Figure 3.
Figure 3.. DNA-PKcs-mediated fork reversal prevents replication-associated DNA damage and is associated with increased tolerance to replication stress.
a) IdU+ cells with >3 53BP1 foci in HCT-116 control or PRKDC depleted clones treated as indicated. Plotted values are the mean of 3 independent biological replicates ±SD. P-values were calculated with the One-way Anova test. Scale bar 10μm. b) IdU+ cells with >4 53BP1 foci in U-2OS cells treated or mock with NU7441 (5μM) as indicated. Plotted values are the mean of 2 independent biological replicates ±SD. P-values were calculated with the One-way Anova test. Scale bar 10μm. c) Clonogenic survival assay in HCT-116 control or PRKDC depleted clones treated with the indicated concentrations of AZD6738 for 12 days. The graph shows relative survival curves from 3 independent biological replicates ±SD. d) Clonogenic survival assay in HCT-116 control or PRKDC depleted clones treated with the indicated concentrations of ETP for 12 days. The graph shows relative survival curves from 3 independent biological replicates ±SD. e) Clonogenic survival assay in U-2OS cells treated with ATRi AZD6738 (0.4μM) with or without the DNA-PKi NU7441 (2.5μM) for 10 days. The graph shows relative survival curves from 3 independent biological replicates ±SD. P-values relative to control cells were calculated with the unpaired two-tailed Student’s t-test. f) Immunoblot analysis of U-2OS and HCT-116 cells treated with ETP (20nM) and NU7441 (5μM).
Figure 4.
Figure 4.. Loss of DNA-PKcs-mediated fork remodeling restores PARPi sensitivity in BRCA2-deficient mammary tumor cells with acquired chemoresistance.
a) General workflow of Mdc1 knock-out in KB2P1.21 (Trp53−/−;Brca2−/−) cells according to Liptay et al. (manuscript submitted). b) DNA fiber analysis in KB2P1.21 NT gRNA and Mdc1−/− cells treated according to the depicted labeling scheme. Dot plot shows the total tract length from 200 individual fibers and the median for each condition scored from 3 biological replicates. **** p<0.001 unpaired One-way Anova test. c) Bar graph showing the percentage of micronucleated KB2P1.21 NT gRNA and Mdc1−/− cells treated, or mock treated, with AZD7648 (2μM) for 48 hours. Plotted values are the mean of 3 independent biological replicates ±SD. P-values were calculated with the unpaired two-tailed Student’s t-test. d) Clonogenic survival assay of KB2P1.21 NT gRNA and Mdc1−/− cells treated, or mock treated, with AZD7648 (2μM) for 10 days. Plotted values are the mean of 3 independent biological replicates ±SD. P-values were calculated with the unpaired two-tailed Student’s t-test. e) Clonogenic survival assay in KB2P1.21 NT gRNA and Mdc1−/− cells treated with the indicated concentrations of Olaparib with or without AZD7648 (2μM) for 12 days. The graph shows relative survival from 3 independent biological replicates ±SD. f) Clonogenic survival assay in RPE1 TP53−/−;BRCA1−/− NT gRNA and MDC1−/− cells treated with 100nM Olaparib with or without AZD7648 (2μM) for 12 days. g) Clonogenic survival assay in KB2P1.21 NT gRNA and Mdc1−/− cells treated with the indicated concentrations of cisplatin with or without AZD7648 (2μM) for 10 days. The graph shows relative survival from 3 independent biological replicates ±SD.
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Comment in

  • The forks guardian.
    Zlotorynski E. Zlotorynski E. Nat Rev Mol Cell Biol. 2022 Nov;23(11):697. doi: 10.1038/s41580-022-00546-y. Nat Rev Mol Cell Biol. 2022. PMID: 36171436 No abstract available.

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