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53BP1 fosters fidelity of homology-directed DNA repair

Abstract

Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1 fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome instability. However, our findings suggest that when challenged by DSBs, BRCA1- and 53BP1-deficient cells may become hypersensitive to, and be eliminated by, RAD52 inhibition.

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Figure 1: Exhaustion of 53BP1 by increasing the load of DSBs suppresses RAD51 accumulation in repair foci during S and G2.
Figure 2: Loss of 53BP1 reduces RAD51 affinity toward damaged chromatin.
Figure 3: Suppression of RAD51 coincides with DSB hyper-resection and RAD52 accumulation in repair foci.
Figure 4: 53BP1 exhaustion suppresses accumulation of the RAD51-loading machinery at damaged chromatin.
Figure 5: 53BP1 exhaustion triggers replacement of RAD51 by RAD52 at sites of DSB repair.
Figure 6: Silencing of 53BP1 attenuates GC and promotes SSA-mediated repair.
Figure 7: Lack of 53BP1 leads to RAD52 dependency in cell survival and is exploited for synthetic viability in BRCA1-deficient cells.
Figure 8: Model of 53BP1's role in HDR-pathway choice.

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Acknowledgements

We are grateful to L. Toledo and K.J. Neelsen for useful suggestions and critical comments on the manuscript. Work in the laboratory of J.L. was supported by grants from the Novo Nordisk Foundation (NNF14CC0001) and the Danish Cancer Society (R72-A4436).

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Contributions

F.O., C.L., and J.L. conceived the project. C.L. and J.L. supervised the study. F.O. carried out high-content microscopy, quantitative image analysis and survival assays. K.S. performed reporter assays, biochemical analyses and survival assays. C.L. performed confocal microscopy. M.A. helped to develop high-content imaging assays. M.-B.R. performed western blots, generated cell lines, and contributed to characterization of the anti-RAD52 antibody. J.L. wrote the manuscript. All authors contributed to conceptual development of the project and manuscript editing.

Corresponding author

Correspondence to Jiri Lukas.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 QIBC defines an intrinsic threshold for 53BP1 accumulation at DSBs.

(a) Schematic depiction of the QIBC workflow to quantitatively interrogate nuclear repair reactions during the cell cycle. A typical experiment consists of three steps. Step 1, exponentially growing cells are exposed to IR and stained for DAPI, cyclin A and a specific genome caretaker. Step 2, images are acquired using automated wide field microscopy and the cells are plotted according to their cell cycle distribution based on DAPI and cyclin A signals (every dot represents a single cell). Step 3, fluorescent signals associated with the genome caretaker are quantified and expressed in a color range (heat map), whose intensity reflects the frequency and magnitude of repair events for every single cell. Typically 1000 cells are analyzed for each condition. Scale bar, 10 μm. Additional technical specification can be found in Online Methods and ref. 24. (b) QIBC of 53BP1 accumulation at DSB sites in U-2-OS cells exposed to increasing dose of IR. The sum of the intensities of 53BP1 foci per nucleus is shown (n=1000 cells for each IR dose). Median levels are depicted in green. A.U., arbitrary units. Green arrow marks the IR dose where 53BP1 accumulation at DSBs reaches its threshold. (c) QIBC of γ-H2AX accumulation at DSB sites in U-2-OS cells exposed to increasing dose of IR. Mean γ-H2AX intensity per nucleus is shown (n=1000 cells for each IR dose). Median levels in red. A.U., arbitrary units.

Supplementary Figure 2 Impaired accumulation of RAD51 at IRIF after 53BP1 exhaustion is evident after preextraction of soluble nuclear protein.

(a) QIBC of RAD51 accumulation at IRIF in U-2-OS cells pulse-labeled with EdU, exposed to the indicated doses of IR, and stained after pre-extraction for DAPI and RAD51 (n=1000 cells for each IR dose). Heat map indicates the sum of the intensities of RAD51 foci per nucleus. A.U., arbitrary units. (b) Schematic depiction of cell cycle gating over DAPI-EdU profile obtained by QIBC (left). Quantification of cell cycle distribution of RAD51 IRIF generated by 1 and 10 Gy respectively (right) under QIBC conditions shown in (a). Median values show the sum of the intensities of RAD51 foci per nucleus (n=300 cells for each IR dose and cell cycle stage). A.U., arbitrary units.

Supplementary Figure 3 Suppression of RAD51 after 53BP1 exhaustion is specific and proceeds with similar kinetics across different cell types.

(a) QIBC of RAD51 accumulation at IRIF in U-2-OS cells treated with control siRNA, exposed to the indicated doses of IR and stained for DAPI, cyclin A and RAD51 (n=1000 cells for each IR dose). The heat map indicates the sum of the intensities of RAD51 foci per nucleus. A.U., arbitrary units. (b) QIBC of RAD51 accumulation at IRIF in U-2-OS cells treated with BRCA1 siRNA and analyzed as in (a). A.U., arbitrary units. (c) QIBC of RAD51 accumulation at IRIF in U-2-OS cells treated with BRCA2 siRNA and analyzed as in (a). A.U., arbitrary units. (d) QIBC of RAD51 accumulation at IRIF in Tert-immortalized RPE cells exposed to the increasing dose range of IR and stained for DAPI, cyclin A and RAD51 (n=1000 cells for each IR dose). The heat map indicates the sum of the intensities of RAD51 foci per nucleus. A.U., arbitrary units.

Supplementary Figure 4 Slowdown of resection by partial knockdown of CtIP restores RAD51 focus formation after 53BP1 exhaustion.

(a) Western blots of total cell lysates of U-2-OS cells treated with increasing doses of CtIP siRNA for 24 h. (b) QIBC of RPA accumulation in repair foci in U-2-OS cells treated with the indicated siRNAs, exposed to the indicated doses of IR and stained after pre-extraction for DAPI and RPA1. Mean RPA intensity per nucleus is shown (n=1000 cells for each IR dose). Median levels are depicted in blue. A.U., arbitrary units. (c) QIBC of RAD51 accumulation at IRIF in U-2-OS cells treated with the indicated siRNAs, exposed to IR (10 Gy), and stained for DAPI, cyclin A and RAD51 (n=1000 cells for each condition). The heat map indicates the sum of the intensities of RAD51 foci per nucleus. The bar chart shows the average sum of RAD51 foci intensity late S and G2 cells depicted in insets (n=300 cells for each condition). A.U., arbitrary units.

Supplementary Figure 5 Characterization of the YFP-RAD52 cell line.

(a) Western blots of total cell lysates of naïve and (YFP-RAD52) variants of U-2-OS cells treated with RAD51 siRNA as indicated. (b) QIBC plot of YFP-RAD52 protein levels in U-2-OS (YFP-RAD52) cells stained with DAPI. Mean intensities of the YFP signal per nucleus are shown. The plot is divided into four intensity layers (left) with representative cells (right). The highlighted intensity layer marks the range of intensities that are similar to the level of endogenous RAD52 and thus delineates cells selected for QIBC analyses throughout this study. (c) QIBC of YFP-RAD52 accumulation at IRIF in U-2-OS (YFP-RAD52) cells treated with control siRNA (top) and CtIP siRNA (bottom), exposed to the indicated doses of IR and stained for DAPI and cyclin A (n=1000 cells for each IR dose). The heat map indicates the sum of the intensities of YFP-RAD52 foci per nucleus. A.U., arbitrary units.

Supplementary Figure 6 Increasing load of DSBs triggers DSB hyper-resection.

(a) Quantification of RPA QIBC shown in Figure 3d. Mean RPA1 intensity per nucleus is shown (n=1000 cells for each IR dose). Median levels are depicted in blue. A.U., arbitrary units. (b) Dot blots of genomic DNA from U-2-OS cells treated with the indicated doses of IR and stained for ssDNA. Primary data (left) and their quantification (right; single data points with mean, n=2 technical replicates) are shown.

Supplementary Figure 7 53BP1 promotes GC and suppresses SSA.

(a) Quantification of the GC reporter assay in U-2-OS cells treated with the indicated siRNAs and transfected with the I-SceI expression plasmid (average ± s.d.; n=3 independent knockdown experiments). (b) The SSA reporter assay in U-2-OS cells treated as in (a). Top panel shows PCR products amplified with the indicated primers (see Fig. 6b for schematic depiction). Bottom panel is quantification of the SSA-specific PCR products (average ± s.d.; n=3 independent knockdown experiments). A.U., arbitrary units. (c) Quantification of the GC reporter assay in U-2-OS cells treated with the indicated siRNAs and transfected with the I-SceI expression plasmid (average ± s.d.; n=3 independent knockdown experiments). (d) The SSA reporter assay in U-2-OS cells treated as in (c). Top panel shows PCR products amplified with the indicated primers as in (b). Bottom panel is quantification of the 0.8 kb SSA-specific PCR products (average ± s.d.; n=3 independent knockdown experiments). A.U., arbitrary units. (e) Clonogenic survival of U-2-OS cells treated with the indicated siRNAs and exposed to the indicated doses of IR (mean values, n=2 technical replicates). (f) Clonogenic survival of U-2-OS cells treated with the indicated siRNAs and exposed to the indicated doses of IR (mean values, n=2 technical replicates). (g) Clonogenic survival of U-2-OS cells treated with the indicated siRNAs and exposed to the indicated doses of IR (mean values, n=2 technical replicates).

Supplementary Figure 8 Specificity of siRNA knockdowns.

Western blots of total cell lysates from U-2-OS cells treated with the indicated siRNAs.

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Ochs, F., Somyajit, K., Altmeyer, M. et al. 53BP1 fosters fidelity of homology-directed DNA repair. Nat Struct Mol Biol 23, 714–721 (2016). https://doi.org/10.1038/nsmb.3251

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