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.

Non-homologous DNA end joining and alternative pathways to double-strand break repair

Key Points

  • Mammalian non-homologous DNA end joining (NHEJ) is the primary pathway for the repair of DNA double-strand breaks (DSBs) throughout the cell cycle, including during S and G2 phases.

  • NHEJ relies on the Ku protein to thread onto each broken DNA end. Ku recruits the enzymes and complexes that are needed to trim (nucleases) or to fill in (polymerases) the ends to make them optimally ligatable by the DNA ligase IV complex.

  • The configuration of the DNA ends determines which of several subpathways of NHEJ is able to join the ends. Because NHEJ is flexible and iterative, any of these subpathways can be used but some pathways are more efficient than others for certain DNA ends.

  • When NHEJ is absent owing to a lack of Ku or the DNA ligase complex, alternative end joining (a-EJ) can join the ends using microhomology (usually >4 bp) and there is often some evidence of templated insertions of substantial length (>10 nucleotides). DNA polymerase θ (Pol θ) is of key importance for a-EJ.

  • The single-strand annealing (SSA) pathway requires further end resection by exonuclease 1 (EXO1), Bloom syndrome RecQ-like helicase (BLM) or DNA replication helicase/nuclease 2 (DNA2) to generate the long 3′ single-strand DNA (ssDNA) tails (>20 nucleotides) that are bound by replication protein A (RPA) to prevent the formation of DNA secondary structures. The 3′ ssDNA tails are annealed by RAD52.

Abstract

DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Overview of non-homologous end joining.
Figure 2: Non-homologous end joining proteins and their known interactions.
Figure 3: The various non-homologous end joining subpathways.
Figure 4: Double-strand break repair pathway choice.
Figure 5: Microhomology length requirement of DNA-end joining pathways.

References

  1. Lieber, M. R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79, 181–211 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Lieber, M. R. & Karanjawala, Z. E. Ageing, repetitive genomes and DNA damage. Nat. Rev. Mol. Cell Biol. 5, 69–75 (2004).

    CAS  PubMed  Google Scholar 

  3. Martin, G. M., Smith, A. C., Ketterer, D. J., Ogburn, C. E. & Disteche, C. M. Increased chromosomal aberrations in first metaphases of cells isolated from the kidneys of aged mice. Isr. J. Med. Sci. 21, 296–301 (1985).

    CAS  PubMed  Google Scholar 

  4. Meek, K., Dang, V. & Lees-Miller, S. P. DNA-PK: the means to justify the ends? Adv. Immunol. 99, 33–58 (2008).

    CAS  PubMed  Google Scholar 

  5. Chang, H. H. Y. et al. Different DNA end configurations dictate which NHEJ components are most important for joining efficiency. J. Biol. Chem. 291, 24377–24389 (2016). This paper describes a biochemical reconstitution of NHEJ using a direct gel assay (without PCR) and all of the major NHEJ components.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Goodarzi, A. A. et al. DNA-PK autophosphorylation facilitates Artemis endonuclease activity. EMBO J. 25, 3880–3889 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Gu, J. et al. DNA-PKcs regulates a single-stranded DNA endonuclease activity of Artemis. DNA Repair (Amst.) 9, 429–437 (2010).

    CAS  Google Scholar 

  8. Chang, H. H., Watanabe, G. & Lieber, M. R. Unifying the DNA end-processing roles of the artemis nuclease: Ku-dependent Artemis resection at blunt DNA ends. J. Biol. Chem. 290, 24036–24050 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Chang, H. H. & Lieber, M. R. Structure-specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex. Nucleic Acids Res. 44, 4991–4997 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Dominski, Z. Nucleases of the metallo-β-lactamase family and their role in DNA and RNA metabolism. Crit. Rev. Biochem. Mol. Biol. 42, 67–93 (2007).

    CAS  PubMed  Google Scholar 

  11. Li, S. et al. Evidence that the DNA endonuclease ARTEMIS also has intrinsic 5′-exonuclease activity. J. Biol. Chem. 289, 7825–7834 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Moshous, D. et al. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 105, 177–186 (2001). This paper reports that Artemis is mutated in human SCID.

    CAS  PubMed  Google Scholar 

  13. Ma, Y., Pannicke, U., Schwarz, K. & Lieber, M. R. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 781–794 (2002). This paper reports the discovery that Artemis is a ss–ds endonuclease (and a 5′ exonuclease).

    CAS  PubMed  Google Scholar 

  14. Niewolik, D. et al. DNA-PKcs dependence of artemis endonucleolytic activity: differences between hairpins and 5′ or 3′ overhangs. J. Biol. Chem. 281, 33900–33909 (2006).

    CAS  PubMed  Google Scholar 

  15. Niewolik, D., Peter, I., Butscher, C. & Schwarz, K. Autoinhibition of the nuclease ARTEMIS is mediated by a physical interaction between its catalytic and C-terminal domains. J. Biol. Chem. 292, 3351–3365 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Malu, S. et al. Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs. J. Exp. Med. 209, 955–963 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. De Ioannes, P., Malu, S., Cortes, P. & Aggarwal, A. K. Structural basis of DNA ligase IV–Artemis interaction in nonhomologous end-joining. Cell Rep. 2, 1505–1512 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Riballo, E. et al. A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to γ-H2AX foci. Mol. Cell 16, 715–724 (2004).

    CAS  PubMed  Google Scholar 

  19. Kurosawa, A. et al. The requirement of Artemis in double-strand break repair depends on the type of DNA damage. DNA Cell Biol. 27, 55–61 (2008).

    CAS  PubMed  Google Scholar 

  20. Kanno, S. et al. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses. EMBO J. 26, 2094–2103 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Li, S. et al. Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF) acts as both a single-stranded DNA endonuclease and a single-stranded DNA 3′ exonuclease and can participate in DNA end joining in a biochemical system. J. Biol. Chem. 286, 36368–36377 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Grundy, G. J. et al. APLF promotes the assembly and activity of non-homologous end joining protein complexes. EMBO J. 32, 112–125 (2013).

    CAS  PubMed  Google Scholar 

  23. Pannunzio, N. R., Li, S., Watanabe, G. & Lieber, M. R. Nonhomologous end joining often uses microhomology: implications for alternative end joining. DNA Repair (Amst.) 17, 74–80 (2014).

    CAS  Google Scholar 

  24. Bebenek, K., Pedersen, L. C. & Kunkel, T. A. Structure–function studies of DNA polymerase lambda. Biochemistry 53, 2781–2792 (2014).

    CAS  PubMed  Google Scholar 

  25. Moon, A. F. et al. Sustained active site rigidity during synthesis by human DNA polymerase μ. Nat. Struct. Mol. Biol. 21, 253–260 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Ma, Y. et al. A biochemically defined system for mammalian nonhomologous DNA end joining. Mol. Cell 16, 701–713 (2004).

    CAS  PubMed  Google Scholar 

  27. Bertocci, B., DeSmet, A., Weill, J.-C. & Reynaud, C. A. Non-overlapping functions of polX family DNA polymerases, pol m, pol l, and TdT, during immunoglobulin V(D)J recombination in vivo. Immunity 25, 31–41 (2006).

    CAS  PubMed  Google Scholar 

  28. Pryor, J. M. et al. Essential role for polymerase specialization in cellular nonhomologous end joining. Proc. Natl Acad. Sci. USA 112, E4537–E4545 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. NickMcElhinny, S. A. & Ramsden, D. A. Polymerase μ is a DNA-directed DNA/RNA polymerase. Mol. Cell. Biol. 23, 2309–2315 (2003).

    CAS  Google Scholar 

  30. NickMcElhinny, S. A. et al. A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. Mol. Cell 19, 357–366 (2005).

    CAS  Google Scholar 

  31. Gu, J. et al. XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps. EMBO J. 26, 1010–1023 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Lieber, M. R. The polymerases for V(D)J recombination. Immunity 25, 7–9 (2006).

    CAS  PubMed  Google Scholar 

  33. Daley, J. M., Laan, R. L. V., Suresh, A. & Wilson, T. E. DNA joint dependence of pol X family polymerase action in nonhomologous end joining. J. Biol. Chem. 280, 29030–29037 (2005).

    CAS  PubMed  Google Scholar 

  34. Daley, J. M., Palmbos, P. L., Wu, D. & Wilson, T. E. Nonhomologous end joining in yeast. Ann. Rev. Genet. 39, 431–451 (2005).

    CAS  PubMed  Google Scholar 

  35. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Daley, J. M. & Wilson, T. E. Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining. DNA Repair (Amst.) 7, 67–76 (2007).

    Google Scholar 

  37. Grawunder, U. et al. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388, 492–495 (1997).

    CAS  PubMed  Google Scholar 

  38. Dai, Y. et al. Nonhomologous end joining and V(D)J recombination require an additional factor. Proc. Natl Acad. Sci. USA 100, 2462–2467 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Ahnesorg, P., Smith, P. & Jackson, S. P. XLF interacts with the XRCC4–DNA ligase IV complex to promote nonhomologous end-joining. Cell 124, 301–313 (2006).

    CAS  PubMed  Google Scholar 

  40. Buck, D. et al. Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell 124, 287–299 (2006). References 39 and 40 describe the discovery of Cernunnos (also known as NEJ1 and XLF).

    CAS  PubMed  Google Scholar 

  41. Brouwer, I. et al. Sliding sleeves of XRCC4–XLF bridge DNA and connect fragments of broken DNA. Nature 535, 566–569 (2016).

    CAS  PubMed  Google Scholar 

  42. Ochi, T. et al. DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair. Science 347, 185–188 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Xing, M. et al. Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway. Nat. Commun. 6, 6233 (2015). References 42 and 43 describe the discovery of PAXX.

    CAS  PubMed  Google Scholar 

  44. Roy, S. et al. XRCC4/XLF interaction is variably required for DNA repair and is not required for ligase IV stimulation. Mol. Cell. Biol. 35, 3017–3028 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Tadi, S. K. et al. PAXX is an accessory c-NHEJ factor that associates with Ku70 and has overlapping functions with XLF. Cell Rep. 17, 541–555 (2016).

    CAS  PubMed  Google Scholar 

  46. Bernstein, N. K. et al. The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase. Mol. Cell 17, 657–670 (2005).

    CAS  PubMed  Google Scholar 

  47. Ahel, I. et al. The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates. Nature 443, 713–716 (2006).

    CAS  PubMed  Google Scholar 

  48. Koch, C. A. et al. Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV. EMBO J. 23, 3874–3885 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Inamdar, K. V. et al. Conversion of phosphoglycolate to phosphate termini on 3′ overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1. J. Biol. Chem. 277, 27162–27168 (2002).

    CAS  PubMed  Google Scholar 

  50. Chen, B. et al. GC/MS methods to quantify the 2-deoxypentos-4-ulose and 3′-phosphoglycolate pathways of 4′ oxidation of 2-deoxyribose in DNA: application to DNA damage produced by gamma radiation and bleomycin. Chem. Res. Toxicol. 20, 1701–1708 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhou, T. et al. Deficiency in 3′-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1). Nucleic Acids Res. 33, 289–297 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Sinden, R. R. & Wells, R. D. DNA structure, mutations and human genetic diseases. Curr. Opin. Biotechnol. 3, 612–622 (1992).

    CAS  PubMed  Google Scholar 

  53. Mimori, T. & Hardin, J. A. Mechanism of interaction between Ku protein and DNA. J. Biol. Chem. 261, 10375–10379 (1986).

    CAS  PubMed  Google Scholar 

  54. Costantini, S., Woodbine, L., Andreoli, L., Jeggo, P. A. & Vindigni, A. Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK. DNA Repair (Amst.) 6, 712–722 (2007).

    CAS  Google Scholar 

  55. Sibanda, B. L. et al. Crystal structure of an Xrcc4–DNA ligase IV complex. Nat. Struct. Biol. 8, 1015–1019 (2001).

    CAS  PubMed  Google Scholar 

  56. Grawunder, U., Zimmer, D., Kulesza, P. & Lieber, M. R. Requirement for an interaction of XRCC4 with DNA ligase IV for wild-type V(D)J recombination and DNA double-strand break repair in vivo. J. Biol. Chem. 273, 24708–24714 (1998).

    CAS  PubMed  Google Scholar 

  57. Grawunder, U., Zimmer, D. & Lieber, M. R. DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains. Curr. Biol. 8, 873–876 (1998).

    CAS  PubMed  Google Scholar 

  58. NickMcElhinny, S. A., Snowden, C. M., McCarville, J. & Ramsden, D. A. Ku recruits the XRCC4–ligase IV complex to DNA ends. Mol. Cell. Biol. 20, 2996–3003 (2000).

    CAS  Google Scholar 

  59. Herrmann, G., Lindahl, T. & Schar, P. S. cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4. EMBO J. 17, 4188–4198 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Westmoreland, J. W., Summers, J. A., Holland, C. L., Resnick, M. A. & Lewis, L. K. Blunt-ended DNA double-strand breaks induced by endonucleases PvuII and EcoRV are poor substrates for repair in Saccharomyces cerevisiae. DNA Repair (Amst.) 9, 617–626 (2010).

    CAS  Google Scholar 

  61. Sibanda, B. L., Chirgadze, D. Y. & Blundell, T. L. Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats. Nature 463, 118–121 (2010). This paper describes the crystal structure of DNA-PKcs at 6.6 Å.

    CAS  PubMed  Google Scholar 

  62. Sibanda, B. L., Chirgadze, D. Y., Ascher, D. B. & Blundell, T. L. DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair. Science 355, 520–524 (2017). This paper describes the crystal structure of DNA-PKcs at 4.3 Å. Although no DNA is present, the 20 kDa portion of the C terminus of Ku80 is present.

    CAS  PubMed  Google Scholar 

  63. West, R. B., Yaneva, M. & Lieber, M. R. Productive and nonproductive complexes of Ku and DNA-PK at DNA termini. Mol. Cell. Biol. 18, 5908–5920 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Spagnolo, L., Rivera-Calzada, A., Pearl, L. H. & Llorca, O. Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Mol. Cell 22, 511–519 (2006).

    CAS  PubMed  Google Scholar 

  65. Ma, Y. et al. The DNA-PKcs phosphorylation sites of human artemis. J. Biol. Chem. 280, 33839–33846 (2005).

    CAS  PubMed  Google Scholar 

  66. Lu, H. et al. A biochemically defined system for coding joint formation in human V(D)J recombination. Mol. Cell 31, 485–497 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Blommers, M. J. et al. Effects of base sequence on the loop folding in DNA hairpins. Biochemistry 28, 7491–7498 (1989).

    CAS  PubMed  Google Scholar 

  68. Povirk, L. F. Processing of damaged DNA ends for double-strand break repair in mammalian cells. ISRN Mol. Biol. 2012, 345805 (2012).

    PubMed Central  Google Scholar 

  69. Henner, W. D., Grunberg, S. M. & Haseltine, W. A. Enzyme action at 3′ termini of ionizing radiation-induced DNA strand breaks. J. Biol. Chem. 258, 15198–15205 (1983).

    CAS  PubMed  Google Scholar 

  70. Henner, W. D., Rodriguez, L. O., Hecht, S. M. & Haseltine, W. A. γ ray induced deoxyribonucleic acid strand breaks. 3′ glycolate termini. J. Biol. Chem. 258, 711–713 (1983).

    CAS  PubMed  Google Scholar 

  71. Valerie, K. & Povirk, L. F. Regulation and mechanisms of mammalian double-strand break repair. Oncogene 22, 5792–5812 (2003).

    CAS  PubMed  Google Scholar 

  72. Povirk, L. F., Zhou, T., Zhou, R., Cowan, M. J. & Yannone, S. M. Processing of 3′-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease. J. Biol. Chem. 282, 3547–3558 (2007).

    CAS  PubMed  Google Scholar 

  73. Yannone, S. M. et al. Coordinate 5′ and 3′ endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase. Nucleic Acids Res. 36, 3354–3365 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Ochi, T., Gu, X. & Blundell, T. L. Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair. Structure 21, 672–679 (2013). References 16, 17 and 74 describe the interaction of Artemis with DNA ligase IV.

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Moon, A. F. et al. Structural insight into the substrate specificity of DNA polymerase mu. Nat. Struc. Mol. Biol. 14, 45–53 (2007).

    CAS  Google Scholar 

  76. Junop, M. S. et al. Crystal structure of the XRCC4 DNA repair protein and implications for end joining. EMBO J. 19, 5962–5970 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Andres, S. N., Modesti, M., Tsai, C. J., Chu, G. & Junop, M. S. Crystal structure of human XLF: a twist in nonhomologous DNA end-joining. Mol. Cell 28, 1093–1101 (2007).

    CAS  PubMed  Google Scholar 

  78. Tsai, C. J., Kim, S. A. & Chu, G. Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends. Proc. Natl Acad. Sci. USA 104, 7851–7856 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Liu, X., Shao, Z., Jiang, W., Lee, B. J. & Zha, S. PAXX promotes KU accumulation at DNA breaks and is essential for end-joining in XLF-deficient mice. Nat. Commun. 8, 13816 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Balmus, G. et al. Synthetic lethality between PAXX and XLF in mammalian development. Genes Dev. 30, 2152–2157 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Lescale, C. et al. Specific roles of XRCC4 paralogs PAXX and XLF during V(D)J recombination. Cell Rep. 16, 2967–2979 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Kumar, V., Alt, F. W. & Frock, R. L. PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line. Proc. Natl Acad. Sci. USA 113, 10619–10624 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Saito, S., Kurosawa, A. & Adachi, N. Mutations in XRCC4 cause primordial dwarfism without causing immunodeficiency. J. Hum. Genet. 61, 679–685 (2016).

    CAS  PubMed  Google Scholar 

  84. Gauss, G. H. & Lieber, M. R. Mechanistic constraints on diversity in human V(D)J recombination. Mol. Cell. Biol. 16, 258–269 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Bhargava, R., Onyango, D. O. & Stark, J. M. Regulation of single-strand annealing and its role in genome maintenance. Trends Genet. 32, 566–575 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Lisby, M. & Rothstein, R. Cell biology of mitotic recombination. Cold Spring Harb. Perspect. Biol. 7, a016535 (2015).

    PubMed  PubMed Central  Google Scholar 

  87. Haber, J. E. Mating-type gene switching in Saccharomyces cerevisiae. Annu. Rev. Genet. 32, 561–599 (1998).

    CAS  PubMed  Google Scholar 

  88. Moynahan, M. E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Mimitou, E. P. & Symington, L. S. Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2. EMBO J. 29, 3358–3369 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Escribano-Diaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872–883 (2013).

    CAS  PubMed  Google Scholar 

  91. Daley, J. M., Niu, H., Miller, A. S. & Sung, P. Biochemical mechanism of DSB end resection and its regulation. DNA Repair (Amst.) 32, 66–74 (2015).

    CAS  Google Scholar 

  92. Xie, A. et al. Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair. Mol. Cell 28, 1045–1057 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  Google Scholar 

  94. Wood, R. D. & Doublie, S. DNA polymerase θ (POLQ), double-strand break repair, and cancer. DNA Repair (Amst.) 44, 22–32 (2016). This paper comprehensively summarizes the current information about Pol θ.

    CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  97. Kent, T., Mateos-Gomez, P. A., Sfeir, A. & Pomerantz, R. T. Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining. eLife 5, e13740 (2016).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  100. Chen, X. et al. Human DNA ligases I, III, and IV-purification and new specific assays for these enzymes. Methods Enzymol. 409, 39–52 (2006).

    CAS  PubMed  Google Scholar 

  101. Han, L. & Yu, K. Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells. J. Exp. Med. 205, 2745–2753 (2008). This paper describes the effect of DNA ligase IV knockout on immunoglobulin class switch recombination in a murine B cell line.

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Masani, S., Han, L., Meek, K. & Yu, K. Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination. Proc. Natl Acad. Sci. USA 113, 1261–1266 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Makharashvili, N. et al. Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. Mol. Cell 54, 1022–1033 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Anand, R., Ranjha, L., Cannavo, E. & Cejka, P. Phosphorylated CtIP Functions as a co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end resection. Mol. Cell 64, 940–950 (2016).

    CAS  PubMed  Google Scholar 

  106. Deshpande, R. A., Lee, J. H., Arora, S. & Paull, T. T. Nbs1 converts the human Mre11/Rad50 nuclease complex into an endo/exonuclease machine specific for protein-DNA adducts. Mol. Cell 64, 593–606 (2016).

    CAS  PubMed  Google Scholar 

  107. Robert, I., Dantzer, F. & Reina-San-Martin, B. Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination. J. Exp. Med. 206, 1047–1056 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. Lange, S. S., Takata, K. & Wood, R. D. DNA polymerases and cancer. Nat. Rev. Cancer 11, 96–110 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  109. Yu, A. M. & McVey, M. Synthesis-dependent microhomology-mediated end joining accounts for multiple types of repair junctions. Nucleic Acids Res. 38, 5706–5717 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Lieber, M. R., Hesse, J. E., Mizuuchi, K. & Gellert, M. Lymphoid V(D)J recombination: nucleotide insertion at signal joints as well as coding joints. Proc. Natl Acad. Sci. USA 85, 8588–8592 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Lieber, M. R. Mechanisms of human lymphoid chromosomal translocations. Nat. Rev. Cancer 16, 387–398 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Murga Penas, E. M. et al. The t(14;18)(q32;q21)/IGH-MALT1 translocation in MALT lymphomas contains templated nucleotide insertions and a major breakpoint region similar to follicular and mantle cell lymphoma. Blood 115, 2214–2219 (2010).

    CAS  PubMed  Google Scholar 

  113. Jaeger, U. et al. Follicular lymphomas BCL-2/IgH junctions contain templated nucleotide insertions: novel insights into the mechanism of t(14;18) translocation. Blood 95, 3520–3529 (2000).

    Google Scholar 

  114. Welzel, N. et al. Templated nucleotide addition and immunoglobulin JH-gene utilization in t(11;14) junctions: implications for the mechanism of translocation and the origin of mantle cell lymphoma. Cancer Res. 61, 1629–1636 (2001). References 113 and 114 were the first to describe template insertions in lymphoid chromosomal translocations.

    CAS  PubMed  Google Scholar 

  115. Pan-Hammarstrom, Q. et al. Impact of DNA ligase IV on nonhomologous end joining pathways during class switch recombination in human cells. J. Exp. Med. 201, 189–194 (2005).

    PubMed  PubMed Central  Google Scholar 

  116. Ceccaldi, R., Rondinelli, B. & D'Andrea, A. D. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 26, 52–64 (2016).

    CAS  PubMed  Google Scholar 

  117. Bhargava, R., Carson, C. R., Lee, G. & Stark, J. M. Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency. Proc. Natl Acad. Sci. USA 114, 728–733 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. Symington, L. S. Mechanism and regulation of DNA end resection in eukaryotes. Crit. Rev. Biochem. Mol. Biol. 51, 195–212 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. Mimitou, E. P. & Symington, L. S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 (2008).

    CAS  PubMed  Google Scholar 

  120. Zhu, Z., Chung, W. H., Shim, E. Y., Lee, S. E. & Ira, G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134, 981–994 (2008). References 119 and 120 describe resection mechanisms for a-EJ and SSA in yeast.

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Sung, P., Krejci, L., Van Komen, S. & Sehorn, M. G. Rad51 recombinase and recombination mediators. J. Biol. Chem. 278, 42729–42732 (2003).

    CAS  PubMed  Google Scholar 

  122. Paques, F. & Haber, J. E. Two pathways for removal of nonhomologous DNA ends during double-strand break repair in S. cerevisiae. Mol. Cell. Biol. 17, 6765–6771 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Pannunzio, N. R., Manthey, G. M. & Bailis, A. M. RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae. DNA Repair (Amst.) 7, 788–800 (2008).

    CAS  Google Scholar 

  124. Ira, G. et al. DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431, 1011–1017 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Jazayeri, A. et al. ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat. Cell Biol. 8, 37–45 (2006).

    CAS  PubMed  Google Scholar 

  126. Huertas, P. & Jackson, S. P. Human CtIP mediates cell cycle control of DNA end resection and double strand break repair. J. Biol. Chem. 284, 9558–9565 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Chen, L., Nievera, C. J., Lee, A. Y. & Wu, X. Cell cycle-dependent complex formation of BRCA1. CtIP. MRN is important for DNA double-strand break repair. J. Biol. Chem. 283, 7713–7720 (2008).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  129. Chen, X. et al. Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation. Nat. Struct. Mol. Biol. 18, 1015–1019 (2011).

    PubMed  PubMed Central  Google Scholar 

  130. Tomimatsu, N. et al. Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice. Nat. Commun. 5, 3561 (2014).

    PubMed  Google Scholar 

  131. Zhou, Y. et al. Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM. Mol. Cell 65, 91–104 (2017).

    CAS  PubMed  Google Scholar 

  132. Beucher, A. et al. ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2. EMBO J. 28, 3413–3427 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Biehs, R. et al. DNA double-strand break resection occurs during non-homologous end joining in G1 but is distinct from resection during homologous recombination. Mol. Cell 65, 671–684 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. Falzon, M., Fewell, J. & Kuff, E. L. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J. Biol. Chem. 268, 10546–10552 (1993).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  136. Boboila, C. et al. Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70. Proc. Natl Acad. Sci. USA 107, 3034–3039 (2010). This is the clearest example of a-EJ in mice with the absence of Ku70 and ligase IV.

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Gostissa, M., Alt, F. W. & Chiarle, R. Mechanisms that promote and suppress chromosomal translocations in lymphocytes. Annu. Rev. Immunol. 29, 319–350 (2011).

    CAS  PubMed  Google Scholar 

  138. Chiarle, R. et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–119 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. de Villartay, J. P. Congenital defects in V(D)J recombination. Br. Med. Bull. 114, 157–167 (2015).

    CAS  PubMed  Google Scholar 

  140. Li, L. et al. A founder mutation in Artemis, an SNM1-like protein, causes SCID in Athabascan-speaking Native Americans. J. Immunol. 168, 6323–6329 (2002).

    CAS  PubMed  Google Scholar 

  141. Volk, T. et al. DCLRE1C (ARTEMIS) mutations causing phenotypes ranging from atypical severe combined immunodeficiency to mere antibody deficiency. Hum. Mol. Genet. 24, 7361–7372 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Woodbine, L., Gennery, A. R. & Jeggo, P. A. The clinical impact of deficiency in DNA non-homologous end-joining. DNA Repair (Amst.) 16, 84–96 (2014).

    CAS  Google Scholar 

  143. de Villartay, J. P. When natural mutants do not fit our expectations: the intriguing case of patients with XRCC4 mutations revealed by whole-exome sequencing. EMBO Mol. Med. 7, 862–864 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. I. Jspeert, H. et al. XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination. Blood 128, 650–659 (2016).

    Google Scholar 

  145. Burg, M.v.d. et al. A DNA-PKcs mutation in a radiosensitive T-B SCID patient inhibits Artemis activation and nonhomologous end-joining. J. Clin. Invest. 119, 91–98 (2009).

    PubMed  Google Scholar 

  146. Woodbine, L. et al. PRKDC mutations in a SCID patient with profound neurological abnormalities. J. Clin. Invest. 123, 2969–2980 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. Mathieu, A. L. et al. PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity. J. Allergy Clin. Immunol. 135, 1578–1588.e5 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. Carvalho, C. M. & Lupski, J. R. Mechanisms underlying structural variant formation in genomic disorders. Nat. Rev. Genet. 17, 224–238 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. Abyzov, A. et al. Analysis of deletion breakpoints from 1,092 humans reveals details of mutation mechanisms. Nat. Commun. 6, 7256 (2015).

    CAS  PubMed  Google Scholar 

  150. Stephens, P. J. et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 144, 27–40 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. Iles, N., Rulten, S., El-Khamisy, S. F. & Caldecott, K. W. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. 27, 3793–3803 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  152. Eustermann, S. et al. Solution structures of the two PBZ domains from human APLF and their interaction with poly(ADP-ribose). Nat. Struct. Mol. Biol. 17, 241–243 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  153. Li, G. Y. et al. Structure and identification of ADP-ribose recognition motifs of APLF and role in the DNA damage response. Proc. Natl Acad. Sci. USA 107, 9129–9134 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  154. Mehrotra, P. V. et al. DNA repair factor APLF is a histone chaperone. Mol. Cell 41, 46–55 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank R. Mosteller for comments on the manuscript. Work in the authors' laboratory is supported by the US National Institutes of Health (NIH) (M.R.L.) and by the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (15H04323 to N.A.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Michael R. Lieber.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information S1 (table)

Abundance of key proteins involved in nonhomologous DNA end joining (NHEJ), alternative end joining (a-EJ), single-strand annealing (SSA) and homologous recombination (HR). (PDF 399 kb)

Supplementary information S2 (figure)

The same DSB can be repaired in many different ways, depending on the order in which NHEJ proteins act. (PDF 200 kb)

PowerPoint slides

Glossary

V(D)J recombination

DNA recombination process during B or T lymphocyte activation in which the antigen receptors variable domain exons are assembled from sub-exonic segments called V, D and J to ultimately generate an immunoglobulin gene or T cell receptor, respectively.

Immunoglobulin heavy chain class switch recombination

The DNA recombination process by which the immunoglobulin heavy chain isotype is changed from producing IgM to producing IgG, IgA or IgE.

Microhomology

One or more base pairs of complementarity at the two DNA ends of a break.

FAT domain

FRAP (FKBP12-rapamycin-associated protein), ATM (ataxia telangiectasia mutated), TRRAP (transformation/transcription domain-associated protein) domain. A structural domain found in phosphatidylinositol 3-kinase-like kinase family members.

Pol X family polymerases

Subfamily of DNA polymerases; based on homology it includes Pol β, Pol μ, Pol λ and terminal deoxynucleotidyltransferase (TdT).

BRCA1 C terminus

(BRCT). Protein domain of approximately 100 aa that binds to phosphoproteins that are often involved in the DNA damage response.

DNA end breathing

Break of the hydrogen bonds between one or more base pairs in the anti-parallel strands of the DNA duplex break.

Templated insertions

Nucleotide additions at a double-strand break repair junction that seem to be direct or inverted repeat copies derived from either strand of either of the two DNA ends.

Chromothripsis

Shattering of chromosomal regions followed by random repair of the DNA fragments in some human neoplasms and inherited disorders.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chang, H., Pannunzio, N., Adachi, N. et al. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol 18, 495–506 (2017). https://doi.org/10.1038/nrm.2017.48

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrm.2017.48

Further reading

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