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53BP1–RIF1–shieldin counteracts DSB resection through CST- and Polα-dependent fill-in

Abstract

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair—which requires a 3′ overhang—and classical non-homologous end joining, which can join unresected ends1,2. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1)3,4,5,6,7,8. When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7–SHLD1–SHLD2–SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors4,6,7,8,9. Here we address the mechanism by which 53BP1–RIF1–shieldin regulates the generation of recombinogenic 3′ overhangs. We report that CTC1–STN1–TEN1 (CST)10, a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)–primase11, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST–Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.

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Fig. 1: Shieldin and CST counteract resection at dysfunctional telomeres.
Fig. 2: 53BP1- and shieldin-dependent localization of CST to dysfunctional telomeres.
Fig. 3: CST localizes to DSBs and represses formation of single-stranded DNA.
Fig. 4: CST and Polα affect the outcome of PARPi in BRCA1-deficient cells.

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Acknowledgements

We thank D. White for mouse husbandry; N. Bosco, R. Karssemeijer, L. Timashev and Y. Doksani for help with CRISPR gene knockouts, image analysis and generating MEFs; and R. Greenberg and C. Price for providing cell lines. The Rockefeller University BioImaging Center provided assistance. This work was supported by grants from the NCI (R35CA210036), ACS and BCRF to T.d.L., a grant from the CIHR (FDN143343) to D.D. and the Banting Postdoctoral fellowship to M.Z.

Author information

Authors and Affiliations

Authors

Contributions

M.Z. initiated this work in the de Lange laboratory. T.K. and F.L. performed resection assays. Z.M. and H.T. performed CST and Polα localization assays and RPA phosphorylation assays. Z.M. performed PARPi and RAD51 assays. Y.G. and T.K. analysed RPA foci. A.B. performed yeast two-hybrid assays. K.T. and H.T. performed co-immunoprecipitation analysis. K.T. and T.d.L. performed telomere fusion assays. D.D. provided information, reagents and advice. T.d.L. conceived the study and wrote the paper with input from all co-authors.

Corresponding author

Correspondence to Titia de Lange.

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D.D. is a founder of, owns equity in and receives funding from Repare Therapeutics.

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Extended data figures and tables

Extended Data Fig. 1 Shieldin and CST counteract telomere hyper-resection.

a-c, Effect of SHLD2 on hyper-resection at telomeres that lack TPP1. a, Immunoblot for CHK1-P, an indicator of TPP1 deletion, in Tpp1f/f MEFs with and without bulk population treatment with a Shld2 sgRNA and/or Cre (representative of three experiments). b, Quantitative analysis of telomere end resection as in Fig. 1c using the cells shown in a. c, Quantification of the extent of resection detected in b, as in Fig. 1d. Mean (centre bars) and s.d. (error bars) from four independent experiments. *P < 0.05, **P < 0.01, two-tailed Welch’s t-test. d, Fluorescence-activated cell sorting (FACS) profiles of the indicated cells incubated with BrdU to measure S phase effects of the Stn1 shRNA. Gating strategy for live cells and singlets is shown below the FACS profiles. Representative of two experiments. e, f, Experiments to verify that the single-stranded DNA signal derives from a 3′ overhang. e, Immunoblot for STN1 and γ-tubulin in Tpp1f/f (Rif1f/+) cells treated with Stn1 shRNA and/or Cre. Representative of two experiments. f, Quantitative assay for telomeric overhangs, as in Fig. 1c. Plugs in the ExoI lanes were treated with the 3′ exonuclease from E. coli. Representative of two experiments.

Extended Data Fig. 2 Hyper-resection at telomeres that lack TPP1 is counteracted by CST and shieldin.

a, Immunoblots showing absence of REV7 and reduction of STN1 expression in the indicated Tpp1f/f and Tpp1f/fRev7−/− MEFs treated with either Ctc1 or Stn1 shRNA. Diminished STN1 expression is used as a proxy for the efficacy of the Ctc1 shRNA. Representative of two experiments. b, Quantitative analysis of telomeric overhangs, as in Fig. 1c. Representative of two experiments. c, Quantification of the effect of Ctc1 and Stn1 shRNA on resection at telomeres that lack TPP1, as in Fig. 1d. Data are obtained from two independent REV7-proficient and two independent REV7-deficient clones (light and dark shading).

Extended Data Fig. 3 No effect of CST depletion on telomere hyper-resection when 53BP1 or RIF1 are absent.

a, SV40LT-immortalized Tpp1f/f53bp1−/− cells were complemented with wild-type 53BP1 or a mutant 53BP1 that lacks the ability to interact with RIF1, treated with a Stn1 shRNA as indicated and analysed by immunoblotting for 53BP1 and STN1. Representative of four experiments. b, Quantitative analysis of telomeric overhangs, as in Fig. 1c. c, Quantification of the resection at telomeres that lack TPP1, in four independent experiments performed as in Fig. 1d. d, Immunoblots showing loss of RIF1 and STN1 in the indicated Tpp1f/fRif1f/+ and Tpp1f/fRif1f/f MEFs treated with Cre (96 h) as indicated, and with or without Stn1 shRNA. Note the diminished levels of RIF1 after Cre, owing to heterozygosity in the Tpp1f/fRif1f/+ cells. e, Quantitative analysis of telomeric overhangs, as in Fig. 1c. f, Quantification of the extent of resection detected, as in e, determined from three independent experiments (indicated by different shades of grey) showing mean (centre bars) and s.d. (error bars). Each experiment involved all indicated samples analysed in parallel. g, h, Experiments to verify that the single-stranded DNA signal derives from a 3′ overhang. g, Immunoblot for STN1 and γ-tubulin in Tpp1f/fRif1f/f cells treated with Stn1 shRNA and/or Cre. Representative of two experiments. h, Quantitative assay for telomeric overhangs, as in Fig. 1c. Plugs in the ExoI lanes were treated with the 3′ exonuclease from E. coli. Representative of two experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Welch's t-test.

Extended Data Fig. 4 SHLD2 counteracts resection at telomeres that lack TRF2.

a, Immunoblots for TRF2 deletion and CHK2 phosphorylation in Trf2f/fLig4−/− MEFs, with and without bulk population treatment with a Shld2 sgRNA and/or Cre. Asterisk, non-specific band. Representative of three experiments. b, Quantitative analysis of telomere end resection, as in Fig. 1c, using the cells shown in a. c, Quantification of the extent of resection detected in b, as in Fig. 1d. Mean (centre bars) and s.d. (error bars) from four independent experiments. *P < 0.05, two-tailed Welch's t-test.

Extended Data Fig. 5 CST interacts with shieldin.

a, Immunoprecipitation of individual mouse CST subunits or the three subunit complex (each subunit bearing a Myc tag) with Flag-tagged mouse SHLD1, co-expressed in 293T cells. Flag-tagged POT1B and POT1A serve as positive and negative controls for CST binding, respectively. Representative of two experiments. b, Two-hybrid analysis of CST-shieldin interaction. Yeast cultures were grown overnight in synthetic complete medium that lacked tryptophan and leucine, to a density of 5 × 107 cells per millilitre. Serial tenfold dilutions were generated and 4 μl of each dilution was spotted on synthetic complete medium that lacked the nutrients tryptophan, leucine, adenine and histidine, and contained 3-aminotriazole (3-AT), as indicated. Plates were then incubated for 5 days at 30 °C before imaging. Representative of three experiments.

Extended Data Fig. 6 Localization of CST and Polα to DSBs.

a, Quantification of HA–STN1 localization to DSBs induced by FOKI, as in Fig. 3e. Mean (centre bars) and s.d. (error bars) from 4–6 independent experiments, with >80 induced nuclei for each condition in each experiment. b, Immunofluorescence for endogenous Polα in FOKI–LacI U2OS cells in S phase and after RO3306 treatment (G2). Dotted lines denote the outline of the nucleus. Representative of two experiments. c, Examples of HA–STN1 and Polα localization at DSBs induced by FOKI in G2-arrested FOKI–LacI U2OS cells (as in Fig. 3f). Representative of three experiments. d, Quantification of co-localization of Polα with DSBs induced by FOKI (as in Fig. 3f). Mean (centre bars) and s.d. (error bars) from three independent experiments, with >80 induced nuclei for each condition in each experiment. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Welch’s t-test.

Extended Data Fig. 7 Effect of STN1 knockdown on the intensity of RPA foci induced by ionizing radiation.

Quantification of Myc–RPA32 intensity per nucleus in the experiments shown in Fig. 3g, h. Medians (centre bars and numbers below) obtained from four independent experiments, with >20 nuclei for each experimental condition in each experiment. Each symbol represents one nucleus. *P < 0.05, ****P < 0.0001, two-tailed Welch's t-test.

Extended Data Fig. 8 Effect of CST and Polα on PARPi treatment of BRCA1-deficient cells.

af, Immunoblots on the MEFs used in Fig. 4a–e to verify the absence of deleted proteins and efficacy of the shRNAs. Reduction in STN1 expression is used as a proxy for the efficacy of the Ctc1 shRNA because no antibody to mouse CTC1 is available. Each immunoblot is representative of three experiments. g, Immunoblots for BRCA1 and STN1 in the cells used in Fig. 4f. Representative of two experiments. hj, Control experiment to assess that cells analysed in Fig. 4f progressed through S phase during treatment with PARPi. h, Experimental timeline, as in Fig. 4f, but with inclusion of BrdU in the medium during treatment with PARPi. i, Example of the assay for the presence of BrdU (immunofluorescence) in metaphases collected after the experimental timeline, as in h. j, Quantification of the BrdU incorporation into metaphase chromosomes, as in i (one experiment with ten metaphases per condition).

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Mirman, Z., Lottersberger, F., Takai, H. et al. 53BP1–RIF1–shieldin counteracts DSB resection through CST- and Polα-dependent fill-in. Nature 560, 112–116 (2018). https://doi.org/10.1038/s41586-018-0324-7

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