Letter | Published:

Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination

Nature volume 518, pages 254257 (12 February 2015) | Download Citation


The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions1,2,3. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.

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Data deposits

Sequence has been deposited with the BioProject database under accession PRJNA269507.


  1. 1.

    et al. The nature of telomere fusion and a definition of the critical telomere length in human cells. Genes Dev. 21, 2495–2508 (2007)

  2. 2.

    & Removal of shelterin reveals the telomere end-protection problem. Science 336, 593–597 (2012)

  3. 3.

    et al. The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres. EMBO J. 29, 2598–2610 (2010)

  4. 4.

    Telomeres at a glance. J. Cell Sci. 125, 4173–4178 (2012)

  5. 5.

    , & TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413 (1998)

  6. 6.

    et al. DNA ligase III as a candidate component of backup pathways of nonhomologous end joining. Cancer Res. 65, 4020–4030 (2005)

  7. 7.

    , , & Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway. J. Mol. Biol. 356, 257–265 (2006)

  8. 8.

    et al. IgH class switching and translocations use a robust non-classical end-joining pathway. Nature 449, 478–482 (2007)

  9. 9.

    et al. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation. PLoS Genet. 7, e1002080 (2011)

  10. 10.

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

  11. 11.

    et al. A two-step mechanism for TRF2-mediated chromosome-end protection. Nature 494, 502–505 (2013)

  12. 12.

    , , , & Low-fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res. 36, 3847–3856 (2008)

  13. 13.

    , & Promiscuous DNA synthesis by human DNA polymerase θ. Nucleic Acids Res. 40, 2611–2622 (2012)

  14. 14.

    , & Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila. PLoS Genet. 6, e1001005 (2010)

  15. 15.

    , & Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954–962 (2014)

  16. 16.

    et al. A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nature Commun. 5, 3216 (2014)

  17. 17.

    et al. Phenotype-based identification of mouse chromosome instability mutants. Genetics 163, 1031–1040 (2003)

  18. 18.

    et al. Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. Nature Struct. Mol. Biol. 20, 317–325 (2013)

  19. 19.

    et al. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways. Nucleic Acids Res. 34, 6170–6182 (2006)

  20. 20.

    et al. Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells. Proc. Natl Acad. Sci. USA 110, 7720–7725 (2013)

  21. 21.

    , , , & Strand-specific postreplicative processing of mammalian telomeres. Science 293, 2462–2465 (2001)

  22. 22.

    et al. Tracking genome engineering outcome at individual DNA breakpoints. Nature Methods 8, 671–676 (2011)

  23. 23.

    , , & Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells. Nucleic Acids Res. 41, e115 (2013)

  24. 24.

    et al. Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers. Nature 451, 1116–1120 (2008)

  25. 25.

    et al. A role for DNA polymerase θ in the timing of DNA replication. Nature Commun. 5, 4285 (2014)

  26. 26.

    , , , & RPA antagonizes microhomology-mediated repair of DNA double-strand breaks. Nature Struct. Mol. Biol. 21, 405–412 (2014)

  27. 27.

    , & RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol. Cell 50, 589–600 (2013)

  28. 28.

    et al. DNA polymerase theta is preferentially expressed in lymphoid tissues and upregulated in human cancers. Int. J. Cancer 109, 9–16 (2004)

  29. 29.

    et al. Overexpression of POLQ confers a poor prognosis in early breast cancer patients. Oncotarget 1, 175–184 (2010)

  30. 30.

    et al. DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability. Proc. Natl Acad. Sci. USA 107, 13390–13395 (2010)

  31. 31.

    et al. Engineered proteins detect spontaneous DNA breakage in human and bacterial cells. Elife 2, e01222 (2013)

  32. 32.

    et al. Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling. PLoS Genet. 10, e1004178 (2014)

  33. 33.

    & Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)

  34. 34.

    et al. Human DNA polymerase θ possesses 5′-dRP lyase activity and functions in single-nucleotide base excision repair in vitro. Nucleic Acids Res. 37, 1868–1877 (2009)

  35. 35.

    & DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nature Cell Biol. 7, 712–718 (2005)

  36. 36.

    , , , & Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal. Science 327, 1657–1661 (2010)

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We thank T. de Lange, R. Greenberg, J. Shay, N. Shima, C. Cazaux and R. Wood for providing key reagents for this study. We are grateful to M. Ji, L. Walton Masters, A. Phillips, A. Pinzaru, F. Yeung, P. Tonzi and J. Wong for technical assistance. We thank S. Kabir and F. Lottersberger for critical reading of the manuscript. This work was supported by a grant from the Breast Cancer Alliance (A.S.), V-foundation (A.S.), Department of Defense Breast Cancer Research Program BC134020 (P.A.M.-G.), Pew-Stewart Scholars Award (A.S.), Pew Scholars Award (E.L.-D.), Novartis Advanced Discovery Institute (E.L.-D.), and a grant from the National Institutes of Health (NIH) AG038677 (E.L.-D.). The A.S. laboratory was supported by start-up funds from the Helen L. and Martin S. Kimmel Center for Stem Cell Biology. The K.M.M. laboratory was supported in part by start-up funds from the University of Texas at Austin and from the Cancer Prevention Research Institute of Texas (CPRIT, R116). K.M.M. is a CPRIT scholar.

Author information


  1. Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU School of Medicine, New York, New York 10016, USA

    • Pedro A. Mateos-Gomez
    •  & Agnel Sfeir
  2. Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin. 2506 Speedway Stop A5000, Austin, Texas 78712, USA

    • Fade Gong
    •  & Kyle M. Miller
  3. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA

    • Nidhi Nair
    •  & Eros Lazzerini-Denchi


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A.S., E.L.-D. and P.A.M.-G. conceived the experimental design. P.A.M.-G. and A.S. performed the experiments and analysed the data. E.L.-D. and N.N. performed telomere-sequencing experiments. F.G. and K.M.M. performed experiments related to Polθ localization at DNA breaks. A.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Agnel Sfeir.

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    Supplementary Information

    This file contains Supplementary Data including sequence analysis of telomere fusions using illumina technology and C‐NHEJ junction sequences.

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