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.

The helicase domain of Polθ counteracts RPA to promote alt-NHEJ

Abstract

Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure–function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR–Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Structure–function analysis reveals the role of Polθ-helicase in alt-NHEJ.
Figure 2: Polθ inhibition increases the efficiency of HR-mediated CRISPR–Cas9 gene targeting.
Figure 3: Polθ-helicase antagonizes RPA to promote DNA annealing and alt-NHEJ in vitro.
Figure 4: Mammalian RPA inhibits alt-NHEJ.
Figure 5: The interplay between Polθ and RPA determines the outcome of DSB repair.

References

  1. Sfeir, A. & Symington, L.S. Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway? Trends Biochem. Sci. 40, 701–714 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Boulton, S.J. & Jackson, S.P. Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J. 15, 5093–5103 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Truong, L.N. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Thyme, S.B. & Schier, A.F. Polq-mediated end joining is essential for surviving DNA double-strand breaks during early zebrafish development. Cell Rep. (2016).

  5. Mateos-Gomez, P.A. et al. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination. Nature 518, 254–257 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ceccaldi, R. et al. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair. Nature 518, 258–262 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wyatt, D.W. et al. Essential roles for polymerase θ-mediated end joining in the repair of chromosome breaks. Mol. Cell 63, 662–673 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wood, R.D. & Doublié, S. DNA polymerase θ (POLQ), double-strand break repair, and cancer. DNA Repair (Amst.) 44, 22–32 (2016).

    Article  CAS  Google Scholar 

  9. Harris, P.V. et al. Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. Mol. Cell. Biol. 16, 5764–5771 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chan, S.H., Yu, A.M. & McVey, M. Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila. PLoS Genet. 6, e1001005 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. van Kregten, M. et al. T-DNA integration in plants results from polymerase-θ-mediated DNA repair. Nat. Plants 2, 16164 (2016).

    Article  CAS  PubMed  Google Scholar 

  12. Koole, W. et al. A polymerase theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat. Commun. 5, 3216 (2014).

    Article  PubMed  CAS  Google Scholar 

  13. Yousefzadeh, M.J. et al. Mechanism of suppression of chromosomal instability by DNA polymerase POLQ. PLoS Genet. 10, e1004654 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Kent, T. et al., Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ. Nat. Struct. Mol. Biol. 22, 230–237 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Higgins, G.S. et al. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown. Cancer Res. 70, 2984–2993 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zelensky, A.N., Schimmel, J., Kool, H., Kanaar, R. & Tijsterman, M. Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA. Nat. Commun. 8, 66 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Saito, S., Maeda, R. & Adachi, N. Dual loss of human POLQ and LIG4 abolishes random integration. Nat. Commun. 8, 16112 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Black, S.J., Kashkina, E., Kent, T. & Pomerantz, R.T. DNA polymerase θ: a unique multifunctional end-joining machine. Genes (Basel) 7, E67 (2016).

    Article  CAS  Google Scholar 

  19. Kent, T. et al. DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides. Nucleic Acids Res. 44, 9381–9392 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Zahn, K.E., Averill, A.M., Aller, P., Wood, R.D. & Doublié, S. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair. Nat. Struct. Mol. Biol. 22, 304–311 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hogg, M., Sauer-Eriksson, A.E. & Johansson, E. Promiscuous DNA synthesis by human DNA polymerase θ. Nucleic Acids Res. 40, 2611–2622 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Seki, M., Marini, F. & Wood, R.D. POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells. Nucleic Acids Res. 31, 6117–6126 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Newman, J.A., Cooper, C.D., Aitkenhead, H. & Gileadi, O. Structure of the helicase domain of DNA polymerase theta reveals a possible role in the microhomology-mediated end-joining pathway. Structure 23, 2319–2330 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Beagan, K. et al. Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair. PLoS Genet. 13, e1006813 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Xie, A., Kwok, A. & Scully, R. Role of mammalian Mre11 in classical and alternative nonhomologous end joining. Nat. Struct. Mol. Biol. 16, 814–818 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rass, E. et al. Role of Mre11 in chromosomal nonhomologous end joining in mammalian cells. Nat. Struct. Mol. Biol. 16, 819–824 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Villarreal, D.D. et al. Microhomology directs diverse DNA break repair pathways and chromosomal translocations. PLoS Genet. 8, e1003026 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Decottignies, A. Microhomology-mediated end joining in fission yeast is repressed by pku70 and relies on genes involved in homologous recombination. Genetics 176, 1403–1415 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Daley, J.M. & Wilson, T.E. Rejoining of DNA double-strand breaks as a function of overhang length. Mol. Cell. Biol. 25, 896–906 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Deng, S.K., Gibb, B., de Almeida, M.J., Greene, E.C. & Symington, L.S. RPA antagonizes microhomology-mediated repair of DNA double-strand breaks. Nat. Struct. Mol. Biol. 21, 405–412 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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 

  32. Shima, N., Munroe, R.J. & Schimenti, J.C. The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm. Mol. Cell. Biol. 24, 10381–10389 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Lemée, F. 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).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bothmer, A. et al. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat. Commun. 8, 13905 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yusufzai, T. & Kadonaga, J.T. HARP is an ATP-driven annealing helicase. Science 322, 748–750 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Audry, J. et al. RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends. EMBO J. 34, 1942–1958 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sugiyama, T., New, J.H. & Kowalczykowski, S.C. DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proc. Natl. Acad. Sci. USA 95, 6049–6054 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bailey, S.M., Goodwin, E.H. & Cornforth, M.N. Strand-specific fluorescence in situ hybridization: the CO-FISH family. Cytogenet. Genome Res. 107, 14–17 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Sfeir, A. & de Lange, T. Removal of shelterin reveals the telomere end-protection problem. Science 336, 593–597 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Doksani, Y. & de Lange, T. Telomere-internal double-strand breaks are repaired by homologous recombination and PARP1/Lig3-dependent end-joining. Cell Reports 17, 1646–1656 (2016).

    Article  CAS  PubMed  Google Scholar 

  44. Lee-Theilen, M., Matthews, A.J., Kelly, D., Zheng, S. & Chaudhuri, J. CtIP promotes microhomology-mediated alternative end joining during class-switch recombination. Nat. Struct. Mol. Biol. 18, 75–79 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Symington, L.S. & Gautier, J. Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271 (2011).

    Article  CAS  PubMed  Google Scholar 

  46. Cejka, P. et al. DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature 467, 112–116 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Niu, H. et al. Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae. Nature 467, 108–111 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen, H., Lisby, M. & Symington, L.S. RPA coordinates DNA end resection and prevents formation of DNA hairpins. Mol. Cell 50, 589–600 (2013).

    Article  CAS  PubMed  Google Scholar 

  49. Audebert, M., Salles, B. & Calsou, P. Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining. J. Biol. Chem. 279, 55117–55126 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Adelman, C.A. et al. HELQ promotes RAD51 paralogue-dependent repair to avert germ cell loss and tumorigenesis. Nature 502, 381–384 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Richards, J.D. et al. Structure of the DNA repair helicase hel308 reveals DNA binding and autoinhibitory domains. J. Biol. Chem. 283, 5118–5126 (2008).

    Article  CAS  PubMed  Google Scholar 

  52. Maga, G. et al. Human DNA polymerase lambda functionally and physically interacts with proliferating cell nuclear antigen in normal and translesion DNA synthesis. J. Biol. Chem. 277, 48434–48440 (2002).

    Article  CAS  PubMed  Google Scholar 

  53. Yusufzai, T., Kong, X., Yokomori, K. & Kadonaga, J.T. The annealing helicase HARP is recruited to DNA repair sites via an interaction with RPA. Genes Dev. 23, 2400–2404 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Driscoll, R. & Cimprich, K.A. HARPing on about the DNA damage response during replication. Genes Dev. 23, 2359–2365 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Goullet de Rugy, T. et al. Excess Polθ functions in response to replicative stress in homologous recombination-proficient cancer cells. Biol. Open 5, 1485–1492 (2016).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  57. Robertson, E., Bradley, A., Kuehn, M. & Evans, M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445–448 (1986).

    Article  CAS  PubMed  Google Scholar 

  58. Cho, N.W., Dilley, R.L., Lampson, M.A. & Greenberg, R.A. Interchromosomal homology searches drive directional ALT telomere movement and synapsis. Cell 159, 108–121 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Kent, T., Chandramouly, G., McDevitt, S.M., Ozdemir, A.Y. & Pomerantz, R.T. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ. Nat. Struct. Mol. Biol. 22, 230–237 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Greenberg (University of Pennsylvania) and T. de Lange (The Rockefeller University) for providing key reagents. We are grateful to A. Pinzaru and R. Barry for providing comments on the manuscript. This work was supported by grants from Pershing Square Sohn cancer research alliance (A.S.), the V-foundation (A.S.), Pew-Stewart scholars award (A.S.), and the National Institutes of Health award 1R01GM115472-01 (R.T.P.). P.A.M.-G. is supported by a fellowship from the Department of Defense (BC134020). S.K.D. is supported by an award from The Leukemia & Lymphoma Society.

Author information

Authors and Affiliations

Authors

Contributions

Experiments were designed by A.S., R.T.P., and P.A.M.-G. P.A.M.-G. and S.K.D. performed in vivo experiments. T.K., S.M., E.K., and T.M.H. performed in vitro experiments. A.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Agnel Sfeir.

Ethics declarations

Competing interests

Agnel Sfeir is a cofounder and shareholder in Repare Therapeutics.

Integrated supplementary information

Supplementary Figure 1 Polθ structure-function analysis

(a) Inactivating polymerase and helicase mutations do not alter the stability of Polθ. Flag epitopes were introduced using CRISPR/Cas9 gene targeting at the C-terminal of Polq in mouse ES cells (CCE) with the following genotype: Polq+/+, PolqΔPol and PolqΔHel. Three independent clones were isolated for each genotype. –ve lysates were obtained from non-targeted Polq+/+ cells. (b) Sequence analysis of the targeted CCE-mES cells with the indicated genotypes. (c) Relative quantification of BRCA1 mRNA in CCE-mES cells treated with shControl and shBRCA1. (d) Western-blot analysis for Flag-Cas9 in cells with the indicated genotype. (e) Examples of junction sequence for Der (6) retrieved from several translocation events and classified according to the presence of insertions, deletions and micro-homology. Junctions that fall in the category of “alt-NHEJ” signature are indicated with a check.

Supplementary Figure 2 The function of Polθ-helicase and polymerase during homologous recombination

(a) Accumulation of Rad51 in response to 1 Gy ionizing radiation of mES (CCE) cells with the indicated genotypes. Four hours post-irradiation, cells were fixed and co-stained with anti-Rad51 and γ-H2AX. Graph represents quantification of cells with > 5 RAD51 foci. (Mean ± SD, n=4 – two independent experiments, each carried out with two independent clonal cell lines, *p<0.05, **p<0.01). Two-tailed Student’s t-test. n.s; not-significant (b) Scheme depicting the method used to investigate the impact of Polθ on the efficiency of gene targeting by HR. (c) Sequence analysis of the CCEs following CRISPR/Cas9 targeting to generate Polq knockout allele. (d) Schematic of the strategy employed to target the Sox2 locus. Primers used for genotyping are indicated. (e) FACS analysis to assess the frequency of CCE cells expressing Zsgreen from the Sox2 locus following CRISPR/Cas9 targeting. Three independent population of cells were identified. (f) Genotyping PCR on sorted cells from (e) with the indicated genotypes. (g) Quantification of CRISPR/Cas9-mediated targeting of Sox2 using Cas9 nuclease in Polq+/+ and Polq−/− mES cells (CCE). As a control, DNA-Pki increases the efficiency of targeting. Bars represent the average of six independent experiments for (−) DNA-PKi and four experiments in the case of (+) DNA-PKi ± S.D. Two-tailed Student’s t-test. n.s; not-significant (h) Quantification of CRISPR targeting with Cas9 nickase of Sox2 in CCE cells with the indicated genotypes. Bars represent mean ± S.D. from six independent experiments in the case of Polq−/− and Polq+/+ and four with PolqΔPol and PolqΔHel mutants. *P < 0.05 and **P < 0.01. Two-tailed Student’s t-test.

Supplementary Figure 3 In vitro analysis of Polθ-helicase function

(a) Coomassie-stained SDS-page gel of purified Polθ–helicase (right) and the trimeric RPA complex (left) (b) Phosphorimager scan of thin layer chromatography plate containing results of Polθ ATPase assay in the presence of ssDNA. (c-d) Polθ–hel promotes DNA annealing independent of ATP in the absence of RPA. (c) Schematic of assay. (d) Non-denaturing gel showing Polθ-hel annealing of complementary ssDNA substrates in the presence and absence of ATP. (e-f) In the absence of RPA, Polθ-helicase stimulates DNA synapsis independently of ATP hydrolysis. (e) Schematic of assay. Increasing amounts of Polq-helicase are mixed with the indicated cy3 and cy5 end-joining model substrates containing 3’ overhangs with 4 nt microhomology in the presence of ATP or AMPPNP. (f) Plot showing relative cy5 fluorescence intensity in the presence of indicated nucleotide and Polθ–hel concentration. (g-h) Polθ–hel performs limited annealing in the presence of E. coli SSB. (g) Schematic of assay. (h) Non-denaturing gel showing Polθ–hel annealing of ssDNA substrates pre-bound by the indicated amounts of RPA and E. coli SSB. Percent annealing indicated. Percent annealing calculated by dividing the intensity of the upper band by the sum of the intensities of the upper and lower bands. (i) Comparison of RPA and E. coli SSB affinity for ssDNA (26 nt). Plots showing fluorescence anisotropy following incubation of the indicated amounts of RPA and E. coli SSB with fluorescein-labeled ssDNA. Since the affinity for ssDNA is similar for RPA and SSB, the helicase is likely to displace RPA due to a specific mechanism rather than lower binding affinity.

Supplementary Figure 4 In vitro analysis of Polθ end-joining activity

(a) Top: Schematic of assay. Polq-pol promotes DNA end-joining in vitro via alt-NHEJ. Following annealing of complementary DNA, the polymerase extends each 3’ minimally paired overhang by using the opposing overhang as a template in trans, resulting in strand displacement synthesis followed by limited terminal transferase activity. Middle: Denaturing gel showing Polθ end-joining product and 48 nt marker. Bottom: Non-denaturing gel showing Polq end-joining product and 55 bp dsDNA marker. Lower molecular weight products are due to Polθ terminal transferase activity on 3’ overhangs. (b) Sequence of the DNA substrates using in end-joining assay. (c) Top: Schematic of control experiment used to validate the end-joining in vitro assay. Schematic adapted from1. Non-denaturing gel showing that Polθ end-joining products are susceptible to EcoRI digestion, which confirms end-joining mechanism. (d) Polθ–helicase promotes alt-NHEJ in the presence of RPA. Left: Schematic of Polθ–pol mediated alt-NHEJ assay in the presence of RPA and Polθ–hel. Non-denaturing gels from two independent experiments showing alt-NHEJ products in reactions with the indicated proteins and ATP (right).

1. Kent, T., Chandramouly, G., McDevitt, S. M., Ozdemir, A. Y. & Pomerantz, R. T. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase theta. Nature structural & molecular biology 22, 230–237, doi:10.1038/nsmb.2961 (2015).

Supplementary Figure 5 Polθ–pol template dependent activity is resistant to RPA binding of ssDNA.

(a) Top: Schematic of the assay. Bottom: Denaturing gel showing Polθ–pol primer-template extension in the presence of the indicated amounts of RPA which were pre-incubated with the primer-template. Black asterisk marks template-independent extension (terminal transferase activity) of duplexed DNA by Polθ–pol. (b) Top: Schematic of the assay. Bottom: Denaturing gel showing Polθ–pol primer extension on a gapped DNA template in the presence of the indicated amounts of RPA.

Supplementary Figure 6 Investigating the interplay between Polθ-helicase and RPA during alt-NHEJ

(a) Alignment of RPA1 protein sequence from human, mouse, S. cerevisiae and S. pombe. Highlighted is the conserved aspartic acid residue at positions 228, 258, 228, and 223, respectively. (b) Representative metaphase spreads from TRF1F/FTRF2F/FLig4−/−Cre-ERT 2 MEFs, with the indicated shRNA treatment, 96 h after Cre expression. CO-FISH assay was performed using a FITC-OO-(CCCTAA)3 PNA probe (green) and a Tamra-OO-(TTAGGG)3 PNA probe (red). DAPI in blue. Examples of alt-NHEJ mediated fusion are indicated by white arrows. Scale bars, 10 μm. (c) Western-blot analysis of Myc-RPA1, total RPA1 and tubulin in TRF1F/FTRF2F/FLig4−/−Cre-ERT2 MEFs treated with shControl and shRPA1. (d) Western blot analysis for RPA1 in TRF1F/FTRF2F/FLig4−/−Cre-ERT2 treated with different amounts of shRPA1. Knock-down efficiency is quantified with image lab (Biorad). (e) Quantification of telomere fusions in shelterin-free cells treated with shRPA1 as in (d). (f) Western-blot analysis for the depletion of Rad51 in TRF1F/FTRF2F/FLig4−/−Cre-ERT2 MEFs. (g) Quantification of telomere fusion (alt-NHEJ) and T-SCE (HR) in the indicated cells treated with shRNAs against Rad51 or control shRNA. Bars represent mean ± S.D. from three independent experiments. *P < 0.05 and **P < 0.01; two-tailed Student’s t-test. (h) Representative images of U2OS cells expressing an inducible TRF1-FokI-ERT2 (mcherry) and co-transfected with Myc-RPA1-IRES-GFP and Flag-Polq-Helicase-IRES-TdTomato (wt or K121M). The percentage of cells expressing RPA1, and those co-expressing RPA1 and Polq-Helicase was determined prior to induction of TRF1-FokI. Mean ± S.D. from three independent experiments. Scale bars, 100 μm. (i) Western blot analysis of cells with the indicated treatment.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–2 (PDF 8375 kb)

Life Sciences Reporting Summary (PDF 131 kb)

Supplementary Data Set 1

Supplementary Data Set 1. (PDF 27799 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mateos-Gomez, P., Kent, T., Deng, S. et al. The helicase domain of Polθ counteracts RPA to promote alt-NHEJ. Nat Struct Mol Biol 24, 1116–1123 (2017). https://doi.org/10.1038/nsmb.3494

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3494

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing