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Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ


Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break–repair pathway that uses sequence microhomology to recombine broken DNA. Although MMEJ has been implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3′ single-strand DNA overhangs with ≥2 bp of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing, then uses the opposing overhang as a template in trans to stabilize the DNA synapse. Polθ exhibits a preference for DNA containing a 5′-terminal phosphate, similarly to polymerases involved in nonhomologous end-joining. Finally, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ that probably promote DNA synapse formation.

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Figure 1: Polθ promotes microhomology-mediated end-joining in vitro and in vivo.
Figure 2: Polθ uses the opposing overhang as a template in trans to stabilize the DNA synapse.
Figure 3: Template preferences for Polθ MMEJ.
Figure 4: Polθ promotes MMEJ of DNA containing internal microhomology.
Figure 5: Polθ promotes DNA synapse formation and strand annealing separately from its replication function.
Figure 6: Insertion loop 2 promotes microhomology-mediated end-joining, DNA binding and formation of polymerase complexes.
Figure 7: Models of Polθ MMEJ.

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  1. McVey, M. & Lee, S.E. MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. Trends Genet. 24, 529–538 (2008).

    Article  CAS  Google Scholar 

  2. Bennardo, N., Cheng, A., Huang, N. & Stark, J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).

    Article  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  Google Scholar 

  4. Deriano, L. & Roth, D.B. Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage. Annu. Rev. Genet. 47, 433–455 (2013).

    Article  CAS  Google Scholar 

  5. Corneo, B. et al. Rag mutations reveal robust alternative end joining. Nature 449, 483–486 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Ghezraoui, H. et al. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol. Cell 55, 829–842 (2014).

    Article  CAS  Google Scholar 

  8. Jones, R.E. et al. Escape from telomere-driven crisis is DNA ligase III dependent. Cell Reports 8, 1063–1076 (2014).

    Article  CAS  Google Scholar 

  9. Zhang, Y. & Jasin, M. An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway. Nat. Struct. Mol. Biol. 18, 80–84 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. 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  Google Scholar 

  12. 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  Google Scholar 

  13. 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  Google Scholar 

  14. 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  Google Scholar 

  15. Roerink, S.F., van Schendel, R. & Tijsterman, M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954–962 (2014).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  17. 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  Google Scholar 

  18. Hogg, M., Seki, M., Wood, R.D., Doublie, S. & Wallace, S.S. Lesion bypass activity of DNA polymerase theta (POLQ) is an intrinsic property of the pol domain and depends on unique sequence inserts. J. Mol. Biol. 405, 642–652 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Arana, M.E., Seki, M., Wood, R.D., Rogozin, I.B. & Kunkel, T.A. Low-fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res. 36, 3847–3856 (2008).

    Article  CAS  Google Scholar 

  21. Yoon, J.H., Roy Choudhury, J., Park, J., Prakash, S. & Prakash, L. A role for DNA polymerase theta in promoting replication through oxidative DNA lesion, thymine glycol, in human cells. J. Biol. Chem. 289, 13177–13185 (2014).

    Article  CAS  Google Scholar 

  22. 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  Google Scholar 

  23. Gunn, A., Bennardo, N., Cheng, A. & Stark, J.M. Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context. J. Biol. Chem. 286, 42470–42482 (2011).

    Article  CAS  Google Scholar 

  24. Gunn, A. & Stark, J.M. I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. Methods Mol. Biol. 920, 379–391 (2012).

    Article  CAS  Google Scholar 

  25. Seki, M. & Wood, R.D. DNA polymerase theta (POLQ) can extend from mismatches and from bases opposite a (6–4) photoproduct. DNA Repair (Amst.) 7, 119–127 (2008).

    Article  CAS  Google Scholar 

  26. Cannavo, E. & Cejka, P. Sae2 promotes dsDNA endonuclease activity within Mre11–Rad50–Xrs2 to resect DNA breaks. Nature 514, 122–125 (2014).

    Article  CAS  Google Scholar 

  27. Garcia-Diaz, M. et al. A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology. Mol. Cell 13, 561–572 (2004).

    Article  CAS  Google Scholar 

  28. Moon, A.F. et al. The X family portrait: structural insights into biological functions of X family polymerases. DNA Repair (Amst.) 6, 1709–1725 (2007).

    Article  CAS  Google Scholar 

  29. Brissett, N.C. et al. Molecular basis for DNA double-strand break annealing and primer extension by an NHEJ DNA polymerase. Cell Reports 5, 1108–1120 (2013).

    Article  CAS  Google Scholar 

  30. Schwede, T., Kopp, J., Guex, N. & Peitsch, M.C. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385 (2003).

    Article  CAS  Google Scholar 

  31. Wang, W., Wu, E.Y., Hellinga, H.W. & Beese, L.S. Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides. J. Biol. Chem. 287, 28215–28226 (2012).

    Article  CAS  Google Scholar 

  32. Brissett, N.C. et al. Structure of a NHEJ polymerase-mediated DNA synaptic complex. Science 318, 456–459 (2007).

    Article  CAS  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  Google Scholar 

  34. Schwede, T., Kopp, J., Guex, N. & Peitsch, M.C. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385 (2003).

    Article  CAS  Google Scholar 

  35. Wang, W., Wu, E.Y., Hellinga, H.W. & Beese, L.S. Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides. J. Biol. Chem. 287, 28215–28226 (2012).

    Article  CAS  Google Scholar 

  36. Guex, N. & Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).

    Article  CAS  Google Scholar 

  37. DeLano, W.L. Unraveling hot spots in binding interfaces: progress and challenges. Curr. Opin. Struct. Biol. 12, 14–20 (2002).

    Article  CAS  Google Scholar 

  38. Fazlieva, R. et al. Proofreading exonuclease activity of human DNA polymerase delta and its effects on lesion-bypass DNA synthesis. Nucleic Acids Res. 37, 2854–2866 (2009).

    Article  CAS  Google Scholar 

  39. Acharya, N. et al. Complex formation of yeast Rev1 with DNA polymerase eta. Mol. Cell. Biol. 27, 8401–8408 (2007).

    Article  CAS  Google Scholar 

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Research was funded by US National Institutes of Health grant (4R00CA160648-03) awarded to R.T.P. and Temple University School of Medicine start-up funds to R.T.P. We thank S. Wallace (University of Vermont) for wild-type Polθ and Polθ L2 expression vectors, J. Stark (Beckman Research Institute, City of Hope) for the U2OS cell line EJ2-GFP24 and A.K. Aggarwal (Mount Sinai Hospital) for Polκ.

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Authors and Affiliations



R.T.P., T.K., G.C., S.M.M. and A.Y.O. performed the experiments. R.T.P. designed the experiments and wrote the paper.

Corresponding author

Correspondence to Richard T Pomerantz.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Further analysis of Polθ MMEJ products.

a, Time course of Polθ MMEJ. (Left panels) Non-denaturing gels showing MMEJ time course with indicated pssDNA. (Right) Plot of MMEJ extension products. % MMEJ = intensity of MMEJ products (upper band (*))/total intensity in each lane. b, Restriction analysis of Polθ MMEJ products. pssDNA with half EcoRI site (left). Schematic of assay (middle). Non-denaturing gel showing MMEJ products following incubation with (lane 3) or without (lane 2) EcoRI (right). c, Length analysis of Polθ MMEJ products. pssDNA-4 substrates of different lengths (left). Schematic of assay (middle). Non-denaturing gel showing MMEJ in the presence of 100 nM pssDNA-4A and indicated concentrations of pssDNA-4B.

Supplementary Figure 2 Polθ performs efficient snap-back replication.

a, Models of Polθ extension of ssDNA. (Left) Polθ template independent extension of ssDNA via terminal transferase activity would allow for the incorporation of any dNTP. (Middle) Polθ template dependent extension in cis via ‘snap-back’ replication would limit incorporation to a dNTP (dGTP) complementary to the template sequence (poly-dC). (Right) Polθ template dependent extension in trans via non-homologous end-joining would allow incorporation of a dNTP (dATP) complementary to the opposing template sequence (poly-dT). b, Poly-dC and poly-dT ssDNA templates (top). Denaturing gels showing Polθ extension products in the presence of indicated dNTPs and DNA substrates (bottom). Efficient ssDNA extension exclusively in the presence of dGTP demonstrates Polθ ‘snap-back’ replication activity (right) while ruling out terminal transferase and non-homologous end-joining activities. c, Evidence for ‘snap-back’ replication on non-homo-polynucleotide ssDNA. (Left) Non-denaturing gel showing MMEJ with the indicated substrate and all 4 dNTPs. (Middle) Non-denaturing gel showing Polθ extension in the presence of indicated dNTPs for the indicated times. Preferential incorporation of dCTP and dTTP confirm ‘snap-back’ replication. Models of Polθ ‘snap-back’ replication (right). d, Evidence for ‘snap-back’ replication on telomere-like pssDNA. Non-denaturing gel showing MMEJ with all 4 dNTPs and the indicated pssDNA (left). The major small molecular weight byproduct is generated by ‘snap-back’ replication as indicated by displacement of the short 25 nt strand by Polθ (right). (Right) Non-denaturing gel showing MMEJ on the indicated pssDNA radio-labeled on the 25 nt strand. Displacement of the 25 nt strand (lane 2) and the small size of the majority of Polθ products produced in the left panel (lane 2) indicates ‘snap-back’ replication. Limited extension of the 25 nt strand also indicates ‘snap-back’ replication (lane 4).

Supplementary Figure 3 Control for polymerase activity on a primer template.

Non-denaturing gel showing primer extension in the presence of indicated Pols and primer-template.

Supplementary Figure 4 Polθ promotes MMEJ of pssDNA-containing internal microhomology relatively far from the 3′ ends of overhangs.

pssDNA substrates (left). Non-denaturing gel showing MMEJ in the presence of the indicated pssDNA substrates (middle). Model of MMEJ (right). Orange boxes highlight microhomology. * = 32P, * = MMEJ products.

Supplementary Figure 5 Structural and functional analysis of insertion loop 2.

a, Polθ L2 binds ssDNA. Non-denaturing gels showing EMSA with Polθ WT (left) and Polθ L2 (right) on Cy3 conjugated ssDNA (top). b, Polθ L2 fails to extend ssDNA. Denaturing gel showing a time course of ssDNA extension on the indicated substrate in the presence of Polθ WT (lanes 2–5) and Polθ L2 (lanes 6–9). c, Loop 2 promotes strand annealing. Schematic of annealing assay (left). Non-denaturing gel showing ssDNA annealing in the presence of Polθ WT (lane 4) and Polθ L2 (lane 3). Error bars, s.d. (n = 3 independent experiments). % annealing = (intensity of upper band)/(sum of the intensities of upper and lower bands). d, Loop 2 promotes Polθ dimers and multimers. (Left) Gel filtration profile of Polθ WT (90 kDa). The presence of monomers, dimers and multimers are indicated. (Middle panels) Native gel of Polθ WT (90 kDa; left) and Polθ L2 (83 kDa; right). The presence of a single band demonstrates that Polθ L2 behaves as a monomer (right); Polθ WT migrates as multiple bands, demonstrating complexes (left). (Right panel) SDS gel of Polθ WT (90 kDa; lane 1) and Polθ L2 (83 kDa; lane 2).

Supplementary Figure 6 Structural model of Polθ superimposed on Bacillus Pol I–DNA complex.

a, Superposition of Bacillus Pol I structure (blue; PDB code 4DQQ)31 in complex with primer-template (orange) and Polθ model (grey; residues 1944-2590) assembled by Swiss Model server ( using Bacillus Pol I:DNA structure (PDB code 4DQQ)31 as a template. Loop 2 lies in between palm and thumb domains and is in close proximity to the 3’ terminus of the primer. b, Critical conserved residues involved in polymerase activity (Asp 539, 749) and fidelity (Tyr 600) are closely aligned with homologous residues in Bacillus Pol I, demonstrating the validity of the model. Bacillus Pol I residues indicated in parentheses. c, Loop 2 lies in close proximity to the 3’ terminus of the primer. Orange, DNA. Blue, positively charged amino acids (K, R). Green, hydrophobic amino acids. Red, negatively charged amino acids (D, E)

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

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