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53BP1 nuclear bodies enforce replication timing at under-replicated DNA to limit heritable DNA damage

Nature Cell Biology (2019) | Download Citation

Abstract

Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown. Here, we show that the formation of 53BP1-NBs interrupts the chain of iterative damage intrinsically embedded in UR-DNA. Unlike clastogen-induced 53BP1 foci that are repaired throughout interphase, 53BP1-NBs restrain replication of the embedded genomic loci until late S phase, thus enabling the dedicated RAD52-mediated repair of UR-DNA lesions. The absence or malfunction of 53BP1-NBs causes premature replication of the affected loci, accompanied by genotoxic RAD51-mediated recombination. Thus, through adjusting replication timing and repair pathway choice at under-replicated loci, 53BP1-NBs enable the completion of genome duplication of inherited UR-DNA and prevent the conversion of stochastic under-replications into genome instability.

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Data availability

Numerical and statistical source data for Figs. 1b,c,f,g, 2c, 3b,c,e, 4a,c,d, 5b,d,e, 6a and Supplementary Figs. 1c,d,h, 2a, 4a,f, 5a,e, and 6a,b,d,f,g have been provided in Supplementary Table 1. Primary image data of fixed cells (Figs. 1e, 3d, 4a, 5b,c, 6a and Supplementary Figs. 2a, 3a, and 4b,c,e; 3D maximum intensity projection, 24-bit colour overlay images in TIF format) and uncropped movies with their corresponding single, full-size images of time-series data for single-cell tracking experiments data (Figs. 1d, 2a,b, 3f, 4b and Supplementary Figs. 1e–g, 3b,c, and 5c; AVI movies and 3D maximum intensity projection, 24-bit colour overlay images in TIF format) have been deposited at the European Bioinformatics Institute (EBI) BioStudies database (https://www.ebi.ac.uk/biostudies/) with accession number S-BSST227. Also deposited under the same accession number at the BioStudies database are image source data (uncropped movies and corresponding single, full-size images of time-series data as 3D maximum intensity projection, 24-bit colour overlay images in TIF format) used to generate data for Figs. 1c,f,g, 2c, 3b,e, 4c and Supplementary Figs. 1h, 4a, 5a, and 6a,b). Source data acquired by QIBC (Figs. 1b, 3c, 4a,d, 5b,d,e, 6a and Supplementary Figs. 1c,d, 2a, 4f, 5e, and 6d,f,g), including software guidance to navigate image data, are available from the corresponding authors upon reasonable request.

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Acknowledgements

Research funding in the Lukas Laboratory was provided by the Novo Nordisk Foundation (grant no. NNF14CC0001) and the Lundbeck Foundation (grant no. R264-2017-2819). The HeLa Kyoto cell line was a gift from S. Narumiya. The authors thank Y. Adachi, D. Durocher, R. Kanaar, C. Dinant, R. Strauss and S. Tateishi for sharing reagents for this study. They acknowledge G. Karemore, J. Bulkescher and J. Dreier (Protein Imaging Platform at Novo Nordisk Center for Protein Research) for technical support, and members of J. S. Hoffmann’s group and all members of the Lukas Laboratory for useful suggestions and critical comments on the manuscript.

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Affiliations

  1. Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    • Julian Spies
    • , Claudia Lukas
    • , Kumar Somyajit
    • , Maj-Britt Rask
    • , Jiri Lukas
    •  & Kai John Neelsen

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Contributions

J.S., J.L., C.L. and K.J.N. conceived the project. J.S. designed, performed and analysed all live cell experiments, analysed 53BP1-NB volumes and their PCNA colocalization using Imaris, performed confocal colocalization experiments of 53BP1 with EdU and PCNA, analysed BLM- and RPA-positive anaphase bridges, counted 53BP1-NBs in U2OS and hTERT-RPE1 cells by QIBC, performed western blotting experiments, and recorded all representative confocal immunofluorescene images. K.J.N. performed and analysed QIBC experiments to quantify 53BP1-NBs, measured and analysed the colocalization of 53BP1-NBs with RAD18, RAD51 or RAD52 using Imaris, and analysed SCEs in chromatin bridges. M.-B.R. and K.S. performed western blotting experiments. M.-B.R. and C.L. generated all cell lines used in the study. All authors interpreted data. J.S. designed the figures with the help of K.J.N., K.S. and C.L. K.J.N. and J.L. wrote the paper with the help of J.S. All authors commented on the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Jiri Lukas or Kai John Neelsen.

Integrated supplementary information

  1. Supplementary Figure 1 Replication-coupled dissolution of 53BP1-NBs.

    a, Parental U2OS cells and a U2OS clone stably expressing RFP-53BP1 and GFP-PCNA were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. Uncropped Western blots in Supplementary Fig. 7. b, U2OS cells shown in Fig. 1b immunostained for endogenous PCNA. Cell cycle stages (G1 phase, 5 S-phase quintiles (I-V) and G2 phase) based on DAPI and PCNA intensities quantified by QIBC. Wide-field microscopy images (1-7) show representative PCNA patterns. The assay was performed 3 times with similar results. c, Number of endogenous 53BP1-NBs throughout interphase in U2OS and hTERT-RPE1 cells quantified by QIBC (also see b). Data represents mean ± SD (n = 3 independent experiments). Data points plotted over bars. d, U2OS cells immunostained for endogenous 53BP1 and PCNA and imaged by confocal microscopy. S phase staging based on PCNA patterns. See also Fig. 1e. e, Overlays of representative MIPs of U2OS cells stably expressing GFP-53BP1 and TagRFP PCNA monitored throughout interphase by time-lapse imaging. Scale bar, 12 µm. Arrow indicates a 53BP1-NB. The assay was performed twice with similar results. f, Left, representative MIP images of U2OS cells stably expressing RFP-53BP1 and GFP-PCNA monitored throughout interphase by time-lapse imaging. The outlined cell is analysed on the right. Scale bar, 12 µm. Right, volume of 53BP1-NBs (purple line), nuclear PCNA intensity (green line) and the colocalization of PCNA with 53BP1-NBs (Pearson’s coefficient, heatmap) of the cell on the left. Broken vertical lines mark period of highest PCNA-53BP1 colocalization. The assay was performed once. g, Representative MIP images of a cell analyzed in Fig. 1g. Arrow indicates a replication-coupled dissolution event in late S phase. Scale bar, 12 µm. h, Trajectories of individual 53BP1-NBs from U2OS cells stably expressing RFP-53BP1 and GFP-PCNA and pulsed with 0.2 µM Aphidicolin for 26 hours prior to imaging. Green and purple broken lines indicate colocalization of PCNA and 53BP1; end of the bar marks NB dissolution; vertical broken line separates early-mid from mid-late S phase. The assay was performed once and 51 NBs were analyzed to generate the graph. Statistical source data in Supplementary Table 1.

  2. Supplementary Figure 2 RAD18-NBs form at UR-DNA independently of 53BP1 and reveal replication timing control by 53BP1.

    a, Left panel, representative confocal MIP images of U2OS cells immunostained for endogenous 53BP1 and RAD18. Scale bar, 12 µm. Inset shows x 4 magnification of the indicated area. Right panel, U2OS cells stably expressing GFP-53BP1 were immunostained for GFP and endogenous RAD18, respectively. Colocalization frequencies of RAD18 with 53BP1-NBs were quantified by QIBC throughout interphase in fixed cells. Data represents mean ± SD (n = 3 independent experiments). Individual data points are plotted over bars. See Supplementary Fig. 1b for cell cycle staging. b, U2OS cells stably expressing RFP-53BP1 and GFP-RAD18 were analyzed by immunoblotting with indicated antibodies; loading control: NUDC. Asterisk indicates an unspecific band. The assay was performed twice with similar results. c, U2OS cells stably expressing GFP-RAD18 and TagRFP-PCNA were treated with control and 53BP1 siRNA for 4 days and analyzed by immunoblotting with indicated antibodies; loading control: KAP1. Asterisk indicates an unspecific band. The immunoblot was performed once. See Supplementary Fig. 7 for uncropped Western blots and Supplementary Table 1 for all statistical source data.

  3. Supplementary Figure 3 Inactive 53BP1 triggers premature replication and NB dissolution defects.

    a, Left Panel, U2OS cells stably expressing GFP-53BP1-MFFR and TagRFP-PCNA were depleted from endogenous 53BP1 for 4 days and immunoblotted with indicated antibodies. 53BP1-MFFR lacks the epitope detected by the anti-53BP1 antibody and is instead detected by an anti-GFP antibody; loading control: KAP1. The assay was performed twice with similar results. See Supplementary Fig. 7 for uncropped Western blots. Right panel, representative MIP images of cells as shown on the left immunostained with an anti-GFP antibody and an antibody recognizing the C-terminus of 53BP1, which is absent in the truncation mutant. Scale bar, 12 µm. The immunoblot was performed once. b, Representative MIP images acquired by confocal imaging of a cell analyzed in Fig. 3b. Scale bar, 12 µm. Inset shows x 3 magnification of the indicated area. Arrow indicates a 53BP1-NB. The experiment was performed twice with similar results. c, Representative MIP images of S-phase cells from U2OS cells stably expressing GFP-53BP1-MFFR without depletion of endogenous 53BP1. Cells were treated with a control siRNA for 4 days prior to confocal microscopy. Arrows indicate two late dissolving 53BP1-MFFR-NBs. Scale bar, 12 µm. The assay was performed twice with similar results.

  4. Supplementary Figure 4 Full-length 53BP1 regulates origin firing at 53BP1-NBs and rescues the dissolution defect of 53BP1-MFFR-NBs.

    a, Trajectories of individual 53BP1-MFFR-NBs from cells treated as in Supplementary Fig. 3c. Green and purple broken lines indicate colocalization of PCNA and 53BP1-MFFR and the end of the bar marks the time of NB dissolution. Bars extending into G2 indicate undissolved 53BP1-NBs, vertical broken line separates early-mid from mid-late S phase. The assay was performed once and 51 NBs were analyzed to generate the graph. b, Representative MIP images of S-phase cells from U2OS cells stably expressing GFP-53BP1 and GFP-53BP1-MFFR immunostained for RIF1 and PCNA and imaged by confocal microscopy. Scale bar, 12 µm. Insets show x 4 magnification of the indicated areas. The assay was performed twice with similar results. c, Representative MIP images of U2OS cells stably expressing GFP-53BP1-MFFR, mock depleted or depleted of endogenous 53BP1 for 4 days, and immunostained for RIF1 and GFP. Cells were imaged by confocal microscopy. Scale bar, 12 µm. The assay was performed once. d, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted of endogenous 53BP1 and RIF1 for 4 days and 3 days, respectively, and immunoblotted with indicated antibodies; loading control: KAP1. The immunoblot was performed once. See Supplementary Fig. 7 for uncropped Western blots. e, Representative MIP images of U2OS cells stained for endogenous 53BP1, PCNA and 5-ethynyl-2´-deoxyuridine (EdU) as analyzed in f. Cells were imaged by confocal microscopy. Scale bar, 12 µm. The assay was performed twice with similar results. f, U2OS cells were mock treated or treated 3 hours with 20 µM CDC7 inhibitor prior to 15-minute pulse labelling with EdU and fixation. Cells were stained for endogenous 53BP1 and EdU and imaged by confocal microscopy. S phase staging was based on EdU patterns. The assay was independently performed twice with 355 and 193 cells analysed for replicate #1 and #2, respectively. g, U2OS cells were treated and stained as in f. EdU and DAPI intensities were measured by QIBC. The assay was performed twice with similar results. See Supplementary Table 1 for all statistical source data.

  5. Supplementary Figure 5 RAD52 catalyzes replication-coupled repair of inherited UR-DNA.

    a, Left panel, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted of endogenous 53BP1 and BRCA2 for 4 days and 2 days, respectively, and immunoblotted with indicated antibodies; loading control: KAP1. The assay was performed twice with similar results. Right panel, trajectories of individual 53BP1-NBs from U2OS cells depleted of endogenous 53BP1 and BRCA2 and monitored by confocal time-lapse imaging. Green and purple broken lines indicate colocalization of PCNA and 53BP1 and the end of the bar marks the time of NB dissolution, vertical broken line separates early-mid from mid-late S phase. 39 NBs from one experiment were analyzed to generate the bar graph. b, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted of endogenous 53BP1 and RAD52 for 4 days and 2 days, respectively, and immunoblotted with indicated antibodies; loading control: KAP1. The assay was performed twice with similar results. c, Representative MIP images acquired by confocal imaging of a cell analyzed in Fig. 4c. Scale bar, 12 µm. Arrow indicates a 53BP1-NB. The assay was performed twice with similar results. d, U2OS cells stably expressing GFP-53BP1 were treated with the indicated siRNAs for 3 days and immunoblotted with indicated antibodies; loading control: NUDC. The immunoblot was performed once. e, U2OS stably expressing GFP-53BP1 were treated with indicated siRNAs for 3 days prior to immunostaining for GFP and endogenous RAD52. Colocalization frequency of RAD52 with 53BP1-NBs was quantified by QIBC throughout interphase in fixed cells. Box and whisker plot with center line at median, box limit at 25th/75th centiles, whiskers indicate minimum and maximum, n = 6 independent experiments. Individual data points are plotted over boxes. See also d and Supplementary Fig. 1b for cell cycle staging. See Supplementary Fig. 7 for uncropped Western blots and Supplementary Table 1 for all numerical data.

  6. Supplementary Figure 6 Loss of 53BP1 at UR-DNA triggers aberrant recombination events and chromosome segregation defects.

    a, Left, 53BP1-NB trajectories from U2OS cells depleted of endogenous 53BP1 and REV7 and monitored by confocal time-lapse imaging. Green and purple broken lines indicate colocalization of PCNA and 53BP1 and the end of the bar marks NB dissolution. Bars extending into G2 indicate undissolved 53BP1-NBs, vertical broken line separates early-mid from mid-late S phase in a and b. 46 NBs from one experiment were analyzed to generate the graph. Right, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. b, Left, 53BP1-NB trajectories from U2OS cells depleted of endogenous 53BP1 and RINN1/SHLD3 and monitored by confocal time-lapse imaging. 40 NBs from 2 independent experiments were analyzed to generate the graph. Right, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. c, U2OS cells were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: NUDC. The immunoblot was performed once. d, Frequency of SCE-negative chromatin bridges in U2OS cells in the first and second mitosis after treatment with 0.2 µM Aphidicolin (see Fig. 5a). Data represents mean ± SD (n = 3 independent experiments). e, HeLa cells depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. f, HeLa cells treated with 0.3 µM Aphidicolin as shown in Fig. 5a were immunostained for phospho-H3S10 and BrdU (also see e). Frequency of SCE-positive and SCE-negative chromatin bridges in the first and second mitosis after Aphidicolin treatment. Data represents mean ± SD (n = 4 independent experiments). g, HeLa cells treated with 0.2 µM Aphidicolin as shown in Fig. 5c were immunostained for FANCD2 and RPA (also see e). Frequency of FANCD2-positive and -negative anaphase bridges in the first and second mitosis after Aphidicolin treatment. Data represents mean ± SD (n = 3 independent experiments). Data points plotted over bars. Uncropped Western blots in Supplementary Fig. 7; statistical source data in Supplementary Table 1.

  7. Supplementary Figure 7

    Representative uncropped Western Blots.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7 and legend for Supplementary Table 1.

  2. Reporting Summary

  3. Supplementary Table 1

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https://doi.org/10.1038/s41556-019-0293-6

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