Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mechanism of tandem duplication formation in BRCA1-mutant cells

Nature volume 551, pages 590–595 (2017)Cite this article

Subjects

Abstract

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus–Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: BRCA1 suppresses Tus–Ter-induced GFP−RFP+ repair products.
Figure 2: Tus–Ter-induced GFP−RFP+ repair products are microhomology-mediated tandem duplications.
Figure 3: Candidate mechanisms of Tus–Ter-induced TDs.
Figure 4: A replicative mechanism involving classical non-homologous end joining mediates Tus–Ter-induced TDs.
Figure 5: Solitary DNA ends form at Tus–Ter-stalled forks.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Duxin, J. P. & Walter, J. C. What is the DNA repair defect underlying Fanconi anemia? Curr. Opin. Cell Biol. 37, 49–60 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. D’Andrea, A. D. Susceptibility pathways in Fanconi’s anemia and breast cancer. N. Engl. J. Med. 362, 1909–1919 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  4. Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell. 40, 179–204 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Garcia-Muse, T . & Aguilera, A. Transcription–replication conflicts: how they occur and how they are resolved. Nat. Rev. Mol. Cell Biol. 17, 553–563 (2016)

    Article  CAS  PubMed  Google Scholar 

  6. Carr, A. M. & Lambert, S. Replication stress-induced genome instability: the dark side of replication maintenance by homologous recombination. J. Mol. Biol. 425, 4733–4744 (2013)

    Article  CAS  PubMed  Google Scholar 

  7. Mayle, R. et al. Mus81 and converging forks limit the mutagenicity of replication fork breakage. Science 349, 742–747 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mulcair, M. D. et al. A molecular mousetrap determines polarity of termination of DNA replication in E. coli. Cell 125, 1309–1319 (2006)

    Article  CAS  PubMed  Google Scholar 

  9. Larsen, N. B., Sass, E., Suski, C., Mankouri, H. W. & Hickson, I. D. The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast. Nat. Commun. 5, 3574 (2014)

    Article  ADS  PubMed  CAS  Google Scholar 

  10. Willis, N. A. et al. BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510, 556–559 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Llorente, B., Smith, C. E. & Symington, L. S. Break-induced replication: what is it and what is it for? Cell Cycle 7, 859–864 (2008)

    Article  CAS  PubMed  Google Scholar 

  12. Anand, R. P., Lovett, S. T. & Haber, J. E. Break-induced DNA replication. Cold Spring Harb. Perspect. Biol. 5, a010397 (2013)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Saini, N. et al. Migrating bubble during break-induced replication drives conservative DNA synthesis. Nature 502, 389–392 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Schlacher, K., Wu, H. & Jasin, M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell 22, 106–116 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Long, D. T., Joukov, V., Budzowska, M. & Walter, J. C. BRCA1 promotes unloading of the CMG helicase from a stalled DNA replication fork. Mol. Cell 56, 174–185 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stark, J. M., Pierce, A. J., Oh, J., Pastink, A. & Jasin, M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol. Cell. Biol. 24, 9305–9316 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Pathania, S. et al. BRCA1 is required for postreplication repair after UV-induced DNA damage. Mol. Cell 44, 235–251 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sy, S. M., Huen, M. S. & Chen, J. PALB2 is an integral component of the BRCA complex required for homologous recombination repair. Proc. Natl Acad. Sci. USA 106, 7155–7160 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhao, W. et al. BRCA1–BARD1 promotes RAD51-mediated homologous DNA pairing. Nature 550, 360–365 (2017)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Menghi, F. et al. The tandem duplicator phenotype as a distinct genomic configuration in cancer. Proc. Natl Acad. Sci. USA 113, E2373–E2382 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nik-Zainal, S. et al. Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature 534, 47–54 (2016)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Popova, T. et al. Ovarian cancers harboring inactivating mutations in CDK12 display a distinct genomic instability pattern characterized by large tandem duplications. Cancer Res. 76, 1882–1891 (2016)

    Article  CAS  PubMed  Google Scholar 

  24. Watkins, J., Tutt, A. & Grigoriadis, A. Tandem duplications contribute to not one but two distinct phenotypes. Proc. Natl Acad. Sci. USA 113, E5257–E5258 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Menghi, F. & Liu, E. T. Reply to Watkins et al.: Whole-genome sequencing-based identification of diverse tandem duplicator phenotypes in human cancers. Proc. Natl Acad. Sci. USA 113, E5259–E5260 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chandramouly, G. et al. BRCA1 and CtIP suppress long-tract gene conversion between sister chromatids. Nat. Commun. 4, 2404 (2013)

    Article  ADS  PubMed  Google Scholar 

  27. Polato, F. et al. CtIP-mediated resection is essential for viability and can operate independently of BRCA1. J. Exp. Med. 211, 1027–1036 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nakanishi, K. et al. Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication. Nat. Struct. Mol. Biol. 18, 500–503 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Xue, X., Sung, P. & Zhao, X. Functions and regulation of the multitasking FANCM family of DNA motor proteins. Genes Dev. 29, 1777–1788 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Larsen, N. B. & Hickson, I. D. RecQ helicases: conserved guardians of genomic integrity. Adv. Exp. Med. Biol. 767, 161–184 (2013)

    Article  CAS  PubMed  Google Scholar 

  31. Deans, A. J. & West, S. C. DNA interstrand crosslink repair and cancer. Natl. Rev. 11, 467–480 (2011)

    Article  CAS  Google Scholar 

  32. Morimatsu, M., Donoho, G. & Hasty, P. Cells deleted for Brca2 COOH terminus exhibit hypersensitivity to gamma-radiation and premature senescence. Cancer Res. 58, 3441–3447 (1998)

    CAS  PubMed  Google Scholar 

  33. Hastings, P. J., Ira, G. & Lupski, J. R. A microhomology-mediated break-induced replication model for the origin of human copy number variation. PLoS Genet. 5, e1000327 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Payen, C., Koszul, R., Dujon, B. & Fischer, G. Segmental duplications arise from Pol32-dependent repair of broken forks through two alternative replication-based mechanisms. PLoS Genet. 4, e1000175 (2008)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Bhowmick, R., Minocherhomji, S. & Hickson, I. D. RAD52 facilitates mitotic DNA synthesis following replication stress. Mol. Cell 64, 1117–1126 (2016)

    Article  CAS  PubMed  Google Scholar 

  36. Doe, C. L., Osman, F., Dixon, J. & Whitby, M. C. DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51. Nucleic Acids Res. 32, 5570–5581 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lambert, S. et al. Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange. Mol. Cell 39, 346–359 (2010)

    Article  CAS  PubMed  Google Scholar 

  38. Nguyen, M. O., Jalan, M., Morrow, C. A., Osman, F. & Whitby, M. C. Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse. eLife 4, e04539 (2015)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Neelsen, K. J. & Lopes, M. Replication fork reversal in eukaryotes: from dead end to dynamic response. Nat. Rev. Mol. Cell Biol. 16, 207–220 (2015)

    Article  CAS  PubMed  Google Scholar 

  40. Slack, A., Thornton, P. C., Magner, D. B., Rosenberg, S. M. & Hastings, P. J. On the mechanism of gene amplification induced under stress in Escherichia coli. PLoS Genet. 2, e48 (2006)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Yan, C. T. et al. XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice. Proc. Natl Acad. Sci. USA 103, 7378–7383 (2006)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lee, J. A., Carvalho, C. M. & Lupski, J. R. A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 131, 1235–1247 (2007)

    Article  CAS  PubMed  Google Scholar 

  43. Smith, C. E., Llorente, B. & Symington, L. S. Template switching during break-induced replication. Nature 447, 102–105 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  44. Anand, R. P. et al. Chromosome rearrangements via template switching between diverged repeated sequences. Genes Dev. 28, 2394–2406 (2014)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Sakofsky, C. J. et al. Translesion polymerases drive microhomology-mediated break-induced replication leading to complex chromosomal rearrangements. Mol. Cell 60, 860–872 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Simsek, D. & Jasin, M. Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation. Nat. Struct. Mol. Biol. 17, 410–416 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hartlerode, A. J., Willis, N. A., Rajendran, A., Manis, J. P. & Scully, R. Complex breakpoints and template switching associated with non-canonical termination of homologous recombination in mammalian cells. PLoS Genet. 12, e1006410 (2016)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Arlt, M. F., Ozdemir, A. C., Birkeland, S. R., Wilson, T. E. & Glover, T. W. Hydroxyurea induces de novo copy number variants in human cells. Proc. Natl Acad. Sci. USA 108, 17360–17365 (2011)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  49. Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015)

    Article  CAS  PubMed  Google Scholar 

  50. Hu, J. et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat. Protocols 11, 853–871 (2016)

    Article  CAS  PubMed  Google Scholar 

  51. Davies, H. et al. HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures. Nat. Med. 23, 517–525 (2017)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Welm, B. E., Dijkgraaf, G. J., Bledau, A. S., Welm, A. L. & Werb, Z. Lentiviral transduction of mammary stem cells for analysis of gene function during development and cancer. Cell Stem Cell 2, 90–102 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Puget, N., Knowlton, M. & Scully, R. Molecular analysis of sister chromatid recombination in mammalian cells. DNA Repair (Amst.) 4, 149–161 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Haber, L. Symington, S. Nik-Zainal and P. J. Campbell for discussions. This work was supported by NCI/DFCI SPORE in Breast Cancer Developmental Research Project Award DF/HCC 5 P50 CA 168504-03 (to N.A.W.), ACS postdoctoral research fellowship PF-12-248-01-DMC (to N.A.W.), R01 ES022054 and R01 CA188032-01 (to E.P.H.), NCI grant P30CA034196 and Andrea Branch and David Elliman Cancer Study Fund (to E.T.L.), grants R01CA095175, R01CA217991, CDMRP OC160440 and HeritX funding (to R.S.), a BIDMC-JAX pilot grant and CDMRP grant BC160172 (to R.S. and E.T.L.). F.W.A. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA

    Nicholas A. Willis, Erin E. Duffey, Arvind Panday & Ralph Scully

  2. Howard Hughes Medical Institute and Department of Genetics, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Richard L. Frock & Frederick W. Alt

  3. The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA

    Francesca Menghi & Edison T. Liu

  4. Department of Medicine, Flow Cytometry Core, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA

    Virginia Camacho

  5. The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

    E. Paul Hasty

  6. The Jackson Laboratory, Bar Harbor, Maine, USA

    Edison T. Liu

Authors
  1. Nicholas A. Willis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard L. Frock
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francesca Menghi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Erin E. Duffey
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Arvind Panday
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Virginia Camacho
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E. Paul Hasty
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Edison T. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Frederick W. Alt
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ralph Scully
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.A.W. and R.S. developed the overall experimental plan. N.A.W. performed or participated in all experiments with the exception of cancer genome analysis. N.A.W. and R.S. planned and designed all the experiments, with additional contributions as follows. HTGTS experiments: plan and design: R.L.F. and F.W.A.; execution: R.L.F. and N.A.W. Cancer genome analysis: plan and design: F.M. and E.T.L.; execution: F.M. Analysis of Brca2 mutant cells: plan and design: E.P.H.; execution: N.A.W. Optimization of FACS analysis and FACS sorting protocols: V.C.; execution: N.A.W. and V.C. Construction and characterization of pHIV lentiviral vectors for expression of Xrcc4: N.A.W., E.E.D., A.P. and R.S. N.A.W. and R.S. wrote the manuscript. Individual figure panels were generated by N.A.W., R.L.F., F.M. and R.S.

Corresponding author

Correspondence to Ralph Scully.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks A. Grigoriadis, J. Jonkers, and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 BRCA1 suppresses RAD51-independent Tus–Ter-induced GFP–RFP+ repair outcomes.

a, Repair frequencies in Brca1fl/exon11 and Brca1Δ/exon11 6×Ter-HR reporter cells transfected with Tus or I-SceI and with either control Luciferase siRNA (siLUC) or Brca1 SMARTpool (siBRCA1). Columns represent mean of technical duplicate samples from ten independent experiments (that is, n = 10). Error bars denote s.e.m. Tus-induced HR, Brca1fl/exon11 cells, t-test siBRCA1 versus siLUC: all measurements P < 0.01; Brca1Δ/exon11 cells, siBRCA1 versus siLUC: total HR: P = 0.0470; STGC: P = 0.0003; LTGC: not significant; LTGC/total HR: P < 0.0001; GFP–RFP+: P = 0.0010. I-SceI-induced HR, Brca1fl/exon11 cells, t-test siBRCA1 versus siLUC: all measurements P < 0.05; Brca1Δ/exon11 cells, t-test siBRCA1 versus siLUC: all measurements P < 0.02. b, Representative primary FACS data for Brca1fl/exon11 and Brca1Δ/exon11 6×Ter-HR reporter cells transfected with empty vector, Tus or I-SceI and with siLUC or siBRCA1. Tus-transfected samples reproduced from Fig. 1b. FACS plots produced from pooled data of technical duplicate samples from three independent experiments. Numbers represent percentages. c, RT–qPCR analysis of Brca1 mRNA in siRNA-treated cells. Data normalized to Gapdh and expressed as fold difference from siLUC sample from the same experiment (x = −2ΔΔCt , with ΔΔCt = [Cttarget − CtGapdh] − [CtsiLUC − CtsiGAPDH]). Error bars denote s.d. of ΔCt value (s.d. = √[s.d.target2 + s.d.Gapdh2]). d, Frequencies of GFP–RFP+ events in Brca1fl/exon11 and Brca1Δ/exon11 6×Ter-HR reporter cells transfected with Tus or I-SceI and with siLUC, siBRCA1, or Rad51 SMARTpool (siRAD51). Columns represent mean of technical duplicate samples, n = 5. Error bars denote s.e.m. Tus-induced GFP–RFP+, Brca1fl/exon11 cells, t-test: all comparisons P < 0.05. Tus-induced GFP–RFP+, Brca1Δ/exon11 cells, t-test: all comparisons P < 0.03. Abundance of RAD51 protein in siRNA-treated Brca1fl/exon11 and Brca1Δ/exon11 6×Ter-HR reporter ES cells. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 Examples of breakpoint sequence analysis of Tus–Ter-induced GFP–RFP+ products.

Class 1 and class 2 rearrangements are microhomology-mediated TDs. a, Structure of the GFP–RFP+ class 1 rearrangement marked with red asterisk in Fig. 2. Cartoon elements as in Figs 1 and 2; orange triangle represents 6×Ter array. Right cartoon denote schematic of TD breakpoint. Grey number denote site of Ter-proximal breakpoint relative to Ter array. In this TD clone, this breakpoint is located 333 bp upstream of the first nucleotide of the first Ter site encountered by the rightward replication fork (that is, position −333). Black number denotes number of base pairs of microhomology at the breakpoint (in this clone, microhomology = 2). Grey arrows identify the orientation of the segments of the TD, relative to the reporter. Top text box, the direct sequence of the TD breakpoint. Green bold text denotes fragments of GFP open reading frame (ORF). Red bold letters denote 2-bp microhomology breakpoint. Black text denotes other reporter sequences. Bottom text box, overlay of TD breakpoint ends (green bold for GFP sequences and red bold for 2-bp microhomology breakpoint) on full-length wild-type GFP (grey). b, Structure of the GFP–RFP+ class 2 rearrangement marked with blue asterisk in Fig. 2. Blue letter ‘B’ indicates a BglII site retained within the TD breakpoint. Right cartoon, schematic of TD breakpoint, elements as in a. In this TD clone, the Ter-proximal TD breakpoint is located 8 bp downstream of the first nucleotide of the first Ter site encountered by the rightward replication fork (that is, position +8). Text box, direct sequence of TD breakpoint. Green bold text denotes fragments of GFP ORF. Orange highlighting: 8-bp fragment of first Ter element retained within the TD breakpoint. Red bold letter denotes 1-bp microhomology breakpoint. Blue highlighting denotes BglII site retained within the TD breakpoint. Black text denotes other reporter sequences.

Extended Data Figure 3 Specificity of BRCA1 loss on Tus–Ter-induced TDs.

a, Tus–Ter-induced and I-SceI-induced TD (GFP−RFP+) products in Brca1fl/exon11 or Brca1∆/exon11 6×Ter-HR cells depleted of indicated repair proteins. Induction of repair products was calculated relative to siLUC controls (which therefore score as 1). Data represents mean of between eight and ten independent experiments, each experimental data point collected as technical duplicates (replicates: siBRCA1, n = 10; siBARD1, n = 9; siCtIP, n = 9; siBLM, n = 8; siFANCM, n = 9; siBRCA2, n = 8; siFANCA, n = 9; siFANCD2, n = 10; siRAD51, n = 9; siSLX4, n = 9). Error bars denote s.e.m. b, Tus-induced and I-SceI-induced STGC (GFP+RFP–) products in Brca1fl/exon11 or Brca1∆/exon11 6×Ter-HR cells depleted of repair proteins indicated. Replicates and error bars as in a. c, Representative primary FACS data for Brca1Δ/exon11 6×Ter-HR reporter cells co-transfected with empty vector (EV), Tus or I-SceI expression vectors (as shown) and siRNAs as shown. FACS plots pooled from technical duplicate samples of four independent experiments. Numbers represent percentages. d, RT–qPCR analysis of Blm, Fancm, Brca2, Fanca, Slx4, Ctip and Bard1 mRNA. Data normalized to Gapdh and expressed as a fold difference from siLUC-treated sample from the same experiment (x = −2ΔΔCt , with ΔΔCt = [Cttarget − CtGapdh] − [CtsiLUC − CtsiGAPDH]). Error bars represent the s.d. of the ΔCt value (s.d. = √[s.d.target2 + s.d.Gapdh2]).

Extended Data Figure 4 Tus–Ter-induced TDs in FANCM- or BLM-depleted Brca1Δ/exon11 6×Ter-HR reporter cells.

a, Southern blot analysis of Tus–Ter-induced LTGC and GFP−RFP+ TD products in FANCM or BLM-depleted Brca1Δ/exon11 6×Ter-HR reporter cells (BglII digest, GFP probe). M, molecular mass marker lane. TD breakpoints were identified by PCR product sequencing. b, Repair frequencies in Brca1fl/exon11 and Brca1Δ/exon11 6×Ter-HR reporter cells transfected with siLUC, siFANCM, siBLM or siFANCM plus siBLM in combination. Columns represent mean of technical duplicate samples, n = 7. Error bars denote s.e.m. Tus–Ter-induced total HR, Brca1fl/exon11 cells, t-test: siFANCM versus siLUC and siBLM versus all others P < 0.0001; Brca1∆/exon11 cells, t-test: siBLM or siFANCM plus siBLM versus siLUC: P < 0.005. Tus–Ter-induced STGC, Brca1fl/exon11 cells, t-test: siFANCM versus siLUC and siBLM versus all others P < 0.0010; Brca1∆/exon11 cells, t-test: siFANCM plus siBLM versus siLUC: P = 0.01. Tus–Ter-induced LTGC, Brca1fl/exon11 cells, t-test: siFANCM or siBLM versus siLUC: P < 0.0001; siFANCM+siBLM versus all others P < 0.005; Brca1∆/exon11 cells, t-test: siFANCM or siBLM versus siLUC: P < 0.01; siFANCM plus siBLM versus all others P < 0.03. Tus–Ter-induced ratio of LTGC:total HR, Brca1fl/exon11 cells, t-test: all siFANCM samples versus those with no siFANCM: P < 0.001; Brca1∆/exon11 cells, t-test: all samples versus siLUC: P < 0.002; siFANCM versus siFANCM plus siBLM: P = 0.0420; siBLM versus siFANCM plus siBLM: P = 0.0294. Tus–Ter-induced TD, Brca1∆/exon11 cells, t-test: siFANCM or siBLM versus siLUC: P < 0.002; siFANCM versus siBLM: not significant; siFANCM plus siBLM versus all others: P < 0.0001. I-SceI-induced total HR, Brca1fl/exon11 cells, t-test: siFANCM versus siBLM: P = 0.0265. I-SceI-induced STGC, Brca1fl/exon11 cells, t-test: siFANCM versus siLUC or siBLM: P < 0.05; siBLM versus siFANCM plus siBLM: P = 0.0445. I-SceI-induced LTGC: not significant. I-SceI-induced ratio LTGC:total HR, Brca1fl/exon11 cells, t-test: all samples versus siLUC: P < 0.03; siFANCM versus siFANCM plus siBLM: P = 0.0305; Brca1∆/exon11 cells, t-test: all samples versus siLUC: P < 0.05; siFANCM versus siBLM: P = 0.0245. I-SceI-induced TD, Brca1∆/exon11 cells, t-test: all samples versus siLUC: P < 0.02. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 5 BRCA2 is not a major suppressor of Tus–Ter-induced TDs.

a, GFP–RFP+ products in Brca1fl/exon11 6×Ter-HR cells transfected with siFANCM or siBLM alone or together with siBRCA1, siBARD1, siBRCA2 or siRAD51. Columns represent mean of technical duplicate samples, n = 5. Error bars denote s.e.m. Tus-induced TDs, t-test: siFANCM plus siBRCA1 or siBARD1 versus all other samples: P < 0.01. siBLM plus siBRCA1 or siBARD1 versus all other samples: P < 0.03. I-SceI-induced TDs, t-test: all comparisons not significant. b, GFP–RFP+ products in Brca1fl/exon11 6×Ter-HR cells after depletion of CtIP. Columns represent mean of technical duplicate samples, n = 11. Error bars denote s.e.m. Tus-induced TD t-test: all samples versus siLUC: P < 0.01; siFANCM plus siCtIP versus siCtIP or siFANCM: P < 0.001; siFANCM plus siBRCA1 versus all other siFANCM samples: P < 0.0001; siBLM plus siCtIP versus siBLM: P < 0.0001; siBLM plus siBRCA1 versus all other siBLM samples: P < 0.0001. I-SceI-induced TD t-test: all samples versus siLUC: P < 0.05; siFANCM plus siCtIP versus siCtIP: P = 0.0311; siFANCM plus siBRCA1 versus all other siFANCM samples: P < 0.01; siFANCM plus siCtIP versus siFANCM: not significant; siBLM plus siBRCA1 versus all other siBLM samples: P < 0.01; siBLM plus siCtIP versus siBLM: not significant. c, GFP–RFP+ products in two independently derived Brca2lex1/lex2 single-copy 6×Ter-HR reporter clones transfected with siRNAs as shown. Columns represent mean of technical duplicate samples, n = 8. Error bars denote s.e.m. Clone #3 Tus-induced TD t-test: siFANCM plus siBRCA1 versus all other samples: P < 0.01; siLUC versus siFANCM plus siBRCA2: P = 0.0131; siFANCM versus siFANCM plus siBRCA2: not significant. Clone #56 Tus-induced TD t-test: siFANCM plus siBRCA1 versus all other samples: P < 0.003; siFANCM versus siFANCM plus siBRCA2: not significant. Clone #3 and clone #56 I-SceI-induced TD: not significant. d, RT–qPCR analysis of siRNA-treated Brca2lex1/lex2 6×Ter-HR cells. Data normalized to Gapdh and expressed as a fold difference from siLUC sample (x = −2ΔΔCt, with ΔΔCt = [Cttarget − CtGapdh] − [CtsiLUC − CtsiGAPDH]). Error bars denote s.d. of the ΔCt value (s.d. = √[s.d.target2 + s.d.Gapdh2]). e, Brca2 gene structure in Brca2lex1/lex2 reporter cells. Grey boxes denote Brca2 exons. PCR primers a, b, and c indicated by arrows. neo denotes neomycin-resistance gene. Asterisk denotes partial deletion of exon 26. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 6 Tus–Ter-induced TDs arise by a replicative mechanism involving canonical end-joining.

a, Southern blot analysis of aneuploid TD clones (AseI digest of gDNA, full-length GFP probe). Same data as Fig. 4b. Parental Ter-HR reporter (P) marks size of unaltered reporter. b, Southern blot analysis of 19 reclones of aneuploid TD clones (AseI digest of gDNA, full-length GFP probe) that contained a second reporter copy. M, molecular mass; R, original aneuploid clone. Lanes 3–20, 19 independent re-clones. For parental and TD structure, see Fig. 4b. c, Tus–Ter-induced TDs in FANCM-depleted Xrcc4fl/fl (#8) and Xrcc4Δ/Δ (#11) cells co-transfected with siRNAs shown. Mean of technical duplicates, n = 5. Error bars denote s.e.m. P values from a Student’s t-test apply to #8 and #11 data unless otherwise stated. siFANCM plus siBRCA1 or siFANCM plus siBARD1 versus all other samples: P < 0.02, except for clone #11; siFANCM plus siBRCA1 versus siFANCM plus siRAD51: not significant; siFANCM plus siBRCA1 versus siFANCM plus siBARD1: not significant; siFANCM plus siBRCA2 or siFANCM plus siRAD51 versus siLUC or siFANCM: not significant. d, Tus–Ter-induced TDs in BLM-depleted Xrcc4fl/fl (#8) and Xrcc4Δ/Δ (#11) cells co-transfected with siRNAs shown. Mean of technical duplicates, n = 5. Error bars denote s.e.m. P values from a Student’s t-test apply to both #8 and #11 data unless otherwise stated. siBLM plus siBRCA1 or siBLM plus siBARD1 versus all other samples in clone #8: P < 0.05. In clone #11, siBLM plus siBRCA1 or siBLM plus siBARD1 versus siBLM plus siRAD51 or siBLM plus siBRCA2: not significant; siBLM plus siBRCA1 versus siBLM plus siBARD1: not significant. siBLM plus siBRCA2 or siBLM plus siRAD51 versus: siLUC or siBLM: not significant. e, RAD51 western blot in siRNA-treated #8 and #11 cells. f, RT–qPCR analysis of Fancm, Brca1, Bard1, Blm, and Brca2 mRNA in siRNA-treated #8 and #11 cells. Data normalized to Gapdh and expressed as fold difference from siLUC sample (x = −2ΔΔCt , with ΔΔCt = [Cttarget − CtGapdh] − [CtsiLUC − CtsiGAPDH]). Error denote bars s.d. of ΔCt value (s.d. = √[s.d.target2 + s.d.Gapdh2]). g, RT–qPCR analysis of Brca1, Fancm and Blm mRNA in siRNA-treated Xrcc4Δ/Δ (#11) cells lentivirally transduced with pHIV-EV or pHIV-mXRCC4 (X4). See f for normalization and error bar details. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7 Breakpoint analysis of Tus–Ter-induced TDs.

a, Span of TDs in Brca1Δ/exon11 6×Ter-HR reporter siFANCM (121 independent TDs), siBRCA1 (44 independent TDs), or siBLM (66 independent TDs) treatment groups. b, Microhomology usage at breakpoint of Tus–Ter-induced TDs for Brca1∆/exon11 cells depleted of FANCM, BRCA1 or BLM. Numbers denote total number of breakpoints with microhomology ≤ 5, excluding untemplated insertions. Grey line denotes expected microhomology usage by chance alone. c, Strand preference of mismatch correction in 14 homeologous breakpoints (that is, microhomology with internal mismatches) of Tus–Ter-induced TDs from Brca1Δ/exon11 cells transfected with siRNAs shown. ‘C/T’ indicates C–T mismatch. A TD site (that is, Ter-proximal or upstream) that underwent mismatch correction is noted. d, Template switches associated with six TD breakpoints. Cartoon format as in Extended Data Fig. 2a. Light grey arrows identify orientation of TD segments relative to the parental reporter. Grey numbers denote position of Ter-proximal sites relative to first Ter site encountered by rightward fork. Black numbers denote breakpoint microhomology use (bp). Template switch insertions as shown. e, Distribution of Ter-proximal sites of TD breakpoints in Brca1∆/exon11 cells for each treatment group, relative to first Ter site encountered by rightward fork. 10-bp binned data. Grey area/orange triangles denote 6× Ter array. Bottom, distribution of Ter-proximal TD sites in Brca1Δ/exon11 6×Ter-HR reporter cells transfected with siFANCM, siBRCA1 or siBLM. The source data are identical to that used for histograms in the top panels, but has been re-presented as ‘survival’ curves, scoring the probability that a Ter-proximal TD site will be positioned to the right of the nucleotide in question. Hence, all groups at nucleotide position −800 are at 100% and all reach 0% by position +300. Mantel–Cox log-rank statistical tests between all pairs are not significant. f, Distribution of ‘upstream’ sites of TD breakpoints in Brca1∆/exon11 cells for each treatment group, relative to splice acceptor of RFP exon B. 100-bp binned data.

Extended Data Figure 8 Analysis of TD and HTGTS breakpoints.

a, Microhomology usage in HTGTS (+) end breakpoints for Tus–Ter-induced translocations from Brca1∆/exon11 cells treated with siLUC (655), siFANCM (612), siBRCA1 (548) or siBLM (633) or Brca1fl/exon11 cells treated with siLUC control (636) siFANCM (658), siBRCA1 (403) or siBLM (405) or I-SceI-induced HTGTS breakpoints for Brca1∆/exon11 cells treated with siFANCM (all: 954; +: 506; −: 403). Breakpoints with insertions or with microhomology use >6 were not included in this analysis. Note that HTGTS breakpoints at Tus–Ter are microhomology skewed in comparison to HTGTS breakpoints at I-SceI. b, Comparison of distributions of Ter-proximal TD sites and HTGTS (+) end breakpoint distribution for Brca1Δ/exon11 6×Ter cells treated with siFANCM (679), siBRCA1 (630), or siBLM (724). Mantel–Cox log-rank test for TD versus HTGTS: siFANCM, P < 0.0001; siBRCA1, P < 0.0001; siBLM, P < 0.0001. Gehan–Breslow–Wilcoxon log-rank statistical test: siFANCM TD versus HTGTS, P < 0.0001; siBRCA1 TD versus HTGTS, P < 0.0001; siBLM TD versus HTGTS, P < 0.0001. Right panel, distribution of Tus-induced HTGTS (+) end breakpoint distributions relative to the Ter array in Brca1Δ/exon11 6×Ter cells transfected with siLUC (786). Mantel–Cox log-rank test for HTGTS: siLUC versus siFANCM, P = 0.0171; siLUC versus siBRCA1, P = 0.0003; siLUC versus siBLM, P < 0.0001; siFANCM versus siBRCA1, P = 0.1528; siFANCM versus siBLM, P = 0.0017; siBLM versus siBRCA1, P = 0.1213. Gehan–Breslow–Wilcoxon log-rank test for HTGTS: siLUC versus siFANCM, P = 0.3108; siLUC versus siBRCA1, P = 0.0009; siLUC versus siBLM, P < 0.0001; siFANCM versus siBRCA1, P = 0.0166; siFANCM versus siBLM, P < 0.0001; siBLM versus siBRCA1, P = 0.0751. 6×Ter array indicated by the grey-shaded region. Orange triangles denote individual Ter sites within the 6×Ter array. Nucleotide position 0 represents first nucleotide of first Ter site encountered by the rightward fork. For all Brca1∆/exon11 treatment groups and Brca1fl/exon11 cells depleted of FANCM, each sample group represents pooled data from three independent biological replicates. For all other Brca1fl/exon11 treatment groups, data shown are from two pooled biological replicates.

Extended Data Figure 9 BRCA1 loss in ovarian and breast carcinomas is associated with widespread TDs of approximately 10 kb (group 1 TDs).

a, Analysis of 92 human ovarian carcinoma genomes (available through the Australian Ovarian Cancer Study (AOCS); http://www.aocstudy.org) and 560 breast carcinoma (BC) genomes (available through the Wellcome Trust Sanger Institute; http://cancer.sanger.ac.uk/cosmic), including 163 triple-negative breast cancer (TNBC) genomes. For each dataset, samples are sorted on the x axis based on increasing number of somatic TDs. y axis: log10 of TD span (in kilobases) within each cancer genome, with median marked with circle. Samples featuring a TDP group 1 profile are indicated in orange. Abrogation of BRCA1 and BRCA2 (by germline mutation, somatic mutation or promoter methylation), and of CDK12 (by somatic mutation) is noted according to key. b, Top, exact numbers of samples analysed for each dataset and each genetic/genomic subgroup indicated in boxes, with digits colour-coded according to key in a. Orange boxes denote group 1 TDP; white boxes denote not group 1 TDP. The numbers comprise only samples for which the relevant genetic annotation is available. Bar charts show percentages of cancer samples with abrogation of BRCA1 (red) or BRCA2 (blue) among the two cancer subsets with or without a TDP group 1 profile; P values calculated by Fisher’s exact test. c, Percentages of cancer samples with (orange) or without (grey) a TDP group 1 profile among the entire datasets and the subsets of samples showing abrogation of BRCA1 (B1m) or BRCA2 (B2m); P values calculated by probability mass function.

Extended Data Figure 10 Downregulation of BRCA1 expression is the most prominent and consistent transcriptional feature of ovarian and breast carcinomas associated with TDP group 1 profile.

Box plots comparing expression levels between cancer samples with (orange) or without (grey) a TDP group 1 profile, relative to nine DNA replication/repair genes, for which a role as potential contributors to the widespread TD formation in cancer has been investigated or suggested. Numbers under each dataset represent number of cancers for which expression data are available. P values calculated by Student’s t-test.

Supplementary information

Life Sciences Reporting Summary (PDF 78 kb)

Supplementary Information

This file contains gel source data figures. (PDF 423 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Willis, N., Frock, R., Menghi, F. et al. Mechanism of tandem duplication formation in BRCA1-mutant cells. Nature 551, 590–595 (2017). https://doi.org/10.1038/nature24477

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature24477

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer