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Nuclear ARP2/3 drives DNA break clustering for homology-directed repair

Naturevolume 559pages6166 (2018) | Download Citation

Abstract

DNA double-strand breaks repaired by non-homologous end joining display limited DNA end-processing and chromosomal mobility. By contrast, double-strand breaks undergoing homology-directed repair exhibit extensive processing and enhanced motion. The molecular basis of this movement is unknown. Here, using Xenopus laevis cell-free extracts and mammalian cells, we establish that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. We demonstrate that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete sub-nuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by non-homologous end joining and does not regulate non-homologous end joining. Our findings establish that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.

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Acknowledgements

We thank T. Swayne and E. L. Munteanu from the Confocal and Specialized Microscopy Shared Resource of the Irving Cancer Research Center (ICRC) and the Flow Cytometry Shared Resource of the ICRC at Columbia University (supported by NIH grants #P30-CA013696, #S10-RR025686); J. Stark for the GFP reporter cell lines; G. Legube for the ER-AsiSI U2OS lines and for sharing unpublished data; J. Bear for the ARPC2-LOXP-CreER MTFs; G. Lahav for the RAD52–mCherry U2OS line; R. Dalla-Favera for the RD and CB33 cell lines; J. Lukas for the pEGFP–NLS–RPA32 construct; R. Baer for the I-SceI plasmid; N. Kato and T. Lagache for manuscript comments; and A. Hollar for Metacyte classifier design. This work was supported by the NIH (R35-CA197606 and P01-CA174653 to J.G., RO1-GM099481 to G.G.G., PHS-GM103314 and PHS-GM109824 to B.T.C., and F30-CA217049 to B.R.S.).

Author information

Affiliations

  1. Institute for Cancer Genetics, College of Physicians and Surgeons, Columbia University, New York, NY, USA

    • Benjamin R. Schrank
    • , Tomas Aparicio
    •  & Jean Gautier
  2. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA

    • Yinyin Li
    •  & Brian T. Chait
  3. Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY, USA

    • Wakam Chang
    •  & Gregg G. Gundersen
  4. Department of Biochemistry and Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY, USA

    • Max E. Gottesman
  5. Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, NY, USA

    • Jean Gautier

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Contributions

J.G. and B.R.S conceived the study and wrote the manuscript. B.R.S. conducted the majority of the experiments and data analyses. T.A. and Y.L. performed the mass spectrometry experiments. G.G.G., M.E.G., W.C., B.T.C., T.A. and Y.L. assisted with data analysis and interpretation.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Jean Gautier.

Extended data figures and tables

  1. Extended Data Fig. 1 Actin filament nucleators localize to chromosomal DSBs in Xenopus extracts and mammalian cells.

    a, Enrichment ratios and spectral counts of actin complexes and repair proteins in DSB-containing chromatin are shown by liquid chromatography–mass spectrometry. b, DNA damage-dependent enrichment of actin complexes (+PflMI) following PIKK inhibition (KU55933: ATMi; VE821: ATRi). RPA shown as a marker of DNA damage. c, Quantification of actin complexes in chromatin relative to +PflMI samples. β-actin n = 3 independent experiments, ARPC4 n = 2 independent experiments, CAPZβ n = 3 independent experiments. Mean and s.e.m shown. d, Representative images of WASP foci after NCS treatment in MTFs. e, Quantification of WASP foci (P calculated by two-sided Mann–Whitney test; data shown as mean; DMSO n = 578 nuclei, NCS n = 556 nuclei). f, Co-localization of γH2AX with WASP foci in MTFs. n = 22 nuclei, r = 0.58 ± 0.021, Pearson. Scale bar, 5 μm or as indicated.

  2. Extended Data Fig. 2 CK-689 does not significantly alter movement of foci.

    a, Summary of DSB movement analyses as related to Figs. 3, 6, and Extended Data Fig. 5. b, MSD of RAD52–mCherry foci and RPA–pEGFP–NLS foci treated with DMSO, CK-689 and CK-666. Data shown as mean and weighted s.e.m.; RAD52: CK-689 n = 3,262 foci from 13 nuclei, CK-666 n = 2,143 foci from 12 nuclei, DMSO n = 3,292 foci from 12 nuclei; RPA32: CK-689 n = 790 foci from 13 nuclei, CK-666 n = 823 foci from 12 nuclei, DMSO n = 1,031 foci from 10 nuclei. MSD of RAD52 DMSO also shown in Fig. 6i. MSD of RAD52 CK-689 and CK-666 also shown in Fig. 3g. MSD of RPA CK-689 and CK-666 also shown in Fig. 3k.

  3. Extended Data Fig. 3 ARP2/3 clusters RAD51 foci.

    a, Schematic of clustering events, as defined by the number of paired points that fall within a given radius (arrow, dashed circle). b, Representative cell cycle distribution of U2OS cells following double-thymidine block (out of five independent experiments). c, Representative images of U2OS-AsiSI nuclei showing RAD51 foci. d, RAD51 foci clustering in G2 cells (P calculated by Spatial analysis plug-in; data shown as mean and s.e.m. of Ripley function; DMSO n = 95 nuclei, CK-666 n = 80 nuclei).

  4. Extended Data Fig. 4 WASP and ARP2/3 inactivation do not impair U2OS cell viability, nuclear area, nuclear sphericity or protein expression.

    a, Cell survival after 8 h of treatment with 100 μM CK-666, 50 μM CK-548, or 3 μM wiskostatin. Data shown as mean and s.e.m. n = 4 independent experiments. b, Cell survival after 48 h exposure to increasing concentrations of CK-666. n = 3 independent experiments. Data shown as mean and s.e.m. c, Cell survival after 48 h exposure to increasing concentrations of CK-548. n = 3 independent experiments. Data shown as mean and s.e.m. d, Representative cell-cycle distribution of cells treated with DMSO, 50 μM CK-666, or 25 μM CK-548 for 48 h (out of three independent experiments). e, Analysis of nuclear sphericity. P calculated by Student’s two-tailed t-test; data shown as mean and s.d.; DMSO n = 117 nuclei, CK-666 n = 117 nuclei. NS, not significant. f, Analysis of nuclear area. P calculated by one-way ANOVA with multiple comparisons; data shown as mean and s.d.; DMSO n = 210 nuclei, CK-548 n = 189 nuclei, CK-666 n = 92 nuclei. g, Expression of RAD51, DNA-PKcs, WASP, and ARPC2 validating antibodies used in Fig. 2e–i (single experiment). MEK2 is a loading control. RPA expression levels also shown.

  5. Extended Data Fig. 5 ARP2/3 enhances movement of 53BP1 foci in G2 cells.

    a, Representative U2OS nuclei showing 53BP1–YFP focus traces over 100 min in G1 cells. b, Median cumulative distance travelled by 53BP1–YFP foci in G1 cells. P calculated by two-tailed Mann–Whitney test; CK-689 n = 462 foci from 14 nuclei, CK-666 n = 647 foci from 14 nuclei. NS, not significant. c, MSD of 53BP1–YFP foci in G1 cells. Data shown as mean and weighted s.e.m.; CK-689 n = 926 foci from 14 nuclei, CK-666 n = 1,234 foci from 14 nuclei. Δt, time interval. d, Left, a representative G1 cell with 53BP1–YFP foci. Right, a representative G2 cell with 53BP1–YFP and Rad52–mCherry foci. 53BP1 foci colocalize with RAD52 foci. r = 0.41 ± 0.17, Pearson, n = 5 independent experiments. e, Representative U2OS nuclei showing traces of 53BP1–YFP foci over 100 min in G2 cells. f, MSD of 53BP1–YFP foci. Data shown as mean and weighted s.e.m.; G1 CK-689 n = 926 foci from 14 nuclei, G2 CK-689 n = 1,403 foci from 12 nuclei. G1 CK-689 curve also shown in c. g, MSD of 53BP1–YFP foci in G2 cells. Data shown as mean and weighted s.e.m.; CK-689 n = 1,403 foci from 12 nuclei, CK-666 n = 1,038 foci from 10 nuclei. G2 CK-689 curve also shown in f.

  6. Extended Data Fig. 6 ARP2/3 promotes assembly of actin foci following DNA damage.

    a, Representative U2OS nuclei showing classes of nuclear actin structures following transient transfection with nuclear actin-chromobody TagGFP. b, Percentage of cells with diffuse signal, nuclear actin-cb foci, or rods with or without NCS (DMSO n = 473 cells, NCS n = 473 cells). c, MSD of actin-cb foci and actin-cb rods. Data shown as mean and weighted s.e.m.; actin-cb foci n = 662 foci from 11 nuclei, actin-cb rods n = 161 rods from 5 nuclei. d, Representative images of U2OS nuclei with actin rods or actin-cb foci following CK-666 treatment (n = 5 independent experiments). e, Representative image of a U2OS nucleus with RPA–mCherry and actin-cb foci of n = 3 independent experiments. Arrowheads indicate sites of RPA–mCherry and actin-cb co-localization. f, Expanded image of RPA–mCherry and actin-cb co-localization from e (red box). Traces of RPA and actin-cb foci are shown in red. Yellow and purple circles encompass RPA and actin-cb foci, respectively. n = 3 independent experiments.

  7. Extended Data Fig. 7 WASP and ARP2/3 mediate DSB repair by homology-directed mechanisms

    a, Representative FACS plots of GFP+ cells in the HDR (DR-GFP) assay. b, Western blot shows expression of WASP and N-WASP following WASP siRNA knockdown in whole-cell lysates (single experiment). WASP siRNA 1: 5′-GAGUGGCUGAGUUACUUGC-3′. c, Representative FACS plots of GFP+ cells in the HDR (DR-GFP) assay in WASP and mock-depleted cells. d, Summary of DR-GFP assay with WASP depletion. P calculated by one-way ANOVA with multiple comparisons; data shown as mean and s.e.m; n = 3 independent experiments. HDR efficiency in the presence of DMSO or wiskostatin (Fig. 5a) shown for comparison. e, Representative FACS plots of GFP+ cells in the SSA (SA-GFP) assay. f, Representative FACS plots of GFP+ cells in the MMEJ (EJ2-GFP) assay. g, Representative FACS plots of GFP+ cells in the NHEJ (EJ5-GFP) assay. h, Summary of DR-GFP, SA-GFP, and EJ5-GFP assays with CK-689. P calculated by two-way ANOVA with multiple comparisons; data shown as mean and s.d.; n = 3 independent experiments. NS, not significant.

  8. Extended Data Fig. 8 Actin nucleation regulates HDR in the nucleus and does not require formin-2 activity.

    a, Western blot shows expression of mCherry-tagged actin constructs in U2OS whole-cell lysates (single experiment). b, Summary of DR-GFP (HDR) assay. P calculated by one-way ANOVA with multiple comparisons; data shown as mean and s.e.m; n = 6 independent experiments. c, Western blot shows knockdown of formin-2 following 48 h transfection of mock or sequence-specific siRNA oligos (single experiment). Formin-2 siRNA #2: 5′-CGUGUAAUCAGAAUGCCCA-3′. d, Summary of DR-GFP assay. P calculated by one-way ANOVA with multiple comparisons; data shown as mean and s.e.m; n = 3 independent experiments. e, Mean cell survival after 48 h exposure to increasing concentrations of the formin inhibitor SMIFH2 (n = 3 independent experiments). f, Summary of DR-GFP assay. P calculated by Student’s two-tailed t-test; data shown as mean and s.e.m; n = 3 independent experiments.

  9. Extended Data Fig. 9 B lymphocytes derived from patients with WAS exhibit reduced DSB end-resection.

    a, Representative cell cycle distribution of CB33 and RD lymphocytes derived from healthy individuals following DMSO treatment (left). The percentage of RPA-positive S-phase cells following DMSO treatment was measured by flow cytometry (right). b, Representative cell cycle distribution of lymphocytes bearing the V75M mutation in the WAS gene or a G>A transition at position 5 in intron 6 of the WAS gene (IVS6+5G>A) following DMSO treatment (left). The percentage of RPA-positive S-phase cells following DMSO treatment was measured by flow cytometry (right). c, Representative cell cycle distribution of CB33 and RD lymphocytes following CPT treatment (left). The percentage of RPA-positive S-phase cells following CPT treatment was measured by flow cytometry (right). d, The cell cycle distribution of WAS V75M or WAS IVS6+5G>A lymphocytes following CPT treatment (left). The percentage of RPA-positive S-phase cells following CPT treatment was measured by flow cytometry (right). For all panels, n = 4 independent experiments.

  10. Extended Data Fig. 10 ARP2/3 inactivation confers sensitivity to DSBs induced in S-phase as well as replication-stress-inducing agents.

    a, Control CB33 lymphocytes or lymphocytes bearing a V75M mutation in the WAS gene were treated with CPT for 0, 12, or 24 h. Per cent viability following CPT treatment was assessed by measuring the fraction of annexin V- and PI-negative cells by flow cytometry. b, Summary of CB33 or WAS V75M lymphocyte survival following CPT treatment. P calculated by two-way ANOVA with multiple comparisons; data shown as mean and s.d.; n = 3 independent experiments. c, Clonogenic U2OS cell survival after 12 h of CPT treatment in the presence of DMSO or increasing concentrations of CK-666. Triplicate experiments; data shown as mean and s.d.; n = 2 independent experiments. d, Clonogenic U2OS cell survival after 12 h of aphidicolin treatment in the presence of DMSO or increasing concentrations of CK-666. Triplicate experiments; data shown as mean and s.d.; n = 2 independent experiments. e, Clonogenic U2OS cell survival after olaparib treatment in the presence of DMSO or increasing concentrations of CK-666 for 14 days. Triplicate experiments; data shown as mean and s.e.m.; n = 2 independent experiments. f, DNA damage induces DSBs, which are repaired preferentially by NHEJ in mammalian cells (blue). In S/G2, DSBs may be repaired by HDR (red). All DSBs recruit WASP, but ARP2/3-dependent actin polymerization occurs only at HDR breaks, which become more mobile. Actin polymerization in the vicinity of DSBs generates forces that result in DSB clustering, optimal DNA end resection, formation of RAD51 foci, and HDR.

Supplementary information

  1. Supplementary Figures

    This file contains the source gels. a, Western blots showing recruitment of actin complexes and Mre11 to damaged chromatin, related to Fig. 1a. b, Western blots showing lack of Arpc2 protein in Arpc2-LoxP-CreER MTFs, related to Fig. 3j. c, Western blots showing expression of WT-NLS and R62D-NLS actin constructs in U2OS cells, related to Fig. 5e. d, Western blots showing recruitment of actin complexes and RPA to damaged chromatin, related to Extended Data Fig. 1b. e, Western blots showing expression of DNA-PK, WASP, RPA, Rad51, and Arpc2 in U2OS cells, related to Extended Data Fig. 4g. f, Western blots showing expression of WASP and N-WASP in U2OS cells, related to Extended Data Fig. 7b. g, Western blots showing expression of WT, R62D, WT-NLS, and R62D-NLS actin tagged with mCherry in U2OS cells, related to Extended Data Fig. 8a. h, Western blots showing expression of Formin 2 in U2OS cells, related to Extended Data Fig. 8c

  2. Reporting Summary

  3. Video 1: Movement of DSBs undergoing HDR. a-d, Representative videos of Rad52-mCherry foci clustering in U2OS cells. Images are acquired every 5 min. Experiments were repeated independently three times with similar results. e-f, Representative videos of Rad52 foci movements in the presence of 100 μM CK-666. Experiments were repeated independently three times with similar results. g-h, Representative videos of RPA-pEGFP foci clustering in mouse-tail fibroblasts. Images are acquired every 5 min. Experiments were repeated independently three times with similar results

  4. Video 2: Movement of nuclear actin-cb foci. a-b, Representative videos of actin-cb foci in U2OS cells. Images are acquired every 30 sec or as indicated. Experiments were repeated independently three times with similar results. c-e, Representative videos of actin-cb foci movements in a U2OS cell before (left) or after (right) 1 hr treatment with 100 μM CK-666. Images are acquired every 30 sec. Experiments were repeated independently two times with similar results

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