Cellular and Molecular Life Sciences

, Volume 76, Issue 19, pp 3861–3873 | Cite as

The deubiquitinase OTUD5 regulates Ku80 stability and non-homologous end joining

  • Fangzhou Li
  • Qianqian Sun
  • Kun Liu
  • Haichao Han
  • Ning Lin
  • Zhongyi Cheng
  • Yueming Cai
  • Feng Tian
  • Zebin Mao
  • Tanjun Tong
  • Wenhui ZhaoEmail author
Original Article


The ability of cells to repair DNA double-strand breaks (DSBs) is important for maintaining genome stability and eliminating oncogenic DNA lesions. Two distinct and complementary pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), are employed by mammalian cells to repair DNA DSBs. Each pathway is tightly controlled in response to increased DSBs. The Ku heterodimer has been shown to play a regulatory role in NHEJ repair. Ku80 ubiquitination contributes to the selection of a DSB repair pathway by causing the removal of Ku heterodimers from DSB sites. However, whether Ku80 deubiquitination also plays a role in regulating DSB repair is unknown. To address this question, we performed a comprehensive study of the deubiquitinase specific for Ku80, and our study showed that the deubiquitinase OTUD5 serves as an important regulator of NHEJ repair by increasing the stability of Ku80. Further studies revealed that OTUD5 depletion impaired NHEJ repair, and hence reduced overall DSB repair. Furthermore, OTUD5-depleted cells displayed excess end resection; as a result, HR repair was facilitated by OTUD5 depletion during the S/G2 phase. In summary, our study demonstrates that OTUD5 is a specific deubiquitinase for Ku80 and establishes OTUD5 as an important and positive regulator of NHEJ repair.


XRCC5 DUBA DNA lesion Deubiquitinases library DNA damage response 



We are grateful to Dr. Jiadong Wang for his suggestion and help. We thank Qihua He for her suggestion and help, and Center of Medical and Health Analysis, Peking University, for confocal microscopy. We appreciate the ALENABIO (Xi’an, China) Company ( for the pathological micro-tissues (Cat. No. BC03119a). We deeply appreciate help from Ning Kon for editing our manuscript.

Author contributions

FL and WZ, conceived and designed the study that led to the submission, acquired data, and interpreted the results; FL performed the experiment; QS, KL, HH, QH, ZC, YM, and FT participated in the revision of the manuscript; ZM and TT approved the final version; and WZ was the corresponding author. The authors declare that they have no conflicts of interest associated with the contents of this article.


Wenhui Zhao was supported by the National Natural Science Foundation of China (Grant No. 85141044).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Availability of data and material

All of the data and material in this paper are available upon request.

Supplementary material

18_2019_3094_MOESM1_ESM.pptx (2 mb)
Supplementary material 1 (PPTX 2090 kb)
18_2019_3094_MOESM2_ESM.xlsx (204 kb)
Supplementary material 2 (XLSX 203 kb)
18_2019_3094_MOESM3_ESM.xlsx (21 kb)
Supplementary material 3 (XLSX 20 kb)
18_2019_3094_MOESM4_ESM.xlsx (12 kb)
Supplementary material 4 (XLSX 11 kb)


  1. 1.
    Khanna KK, Jackson SP (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet 27:247–254. CrossRefGoogle Scholar
  2. 2.
    Streffer C (2010) Strong association between cancer and genomic instability. Radiat Environ Biophys 49:125–131. CrossRefGoogle Scholar
  3. 3.
    Lieber MR (2008) The mechanism of human nonhomologous DNA end joining. J Biol Chem 283:1–5. CrossRefGoogle Scholar
  4. 4.
    Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211. CrossRefGoogle Scholar
  5. 5.
    Mazon G, Mimitou EP, Symington LS (2010) SnapShot: homologous recombination in DNA double-strand break repair. Cell 142(646):e641. Google Scholar
  6. 6.
    San Filippo J, Sung P, Klein H (2008) Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 77:229–257. CrossRefGoogle Scholar
  7. 7.
    Chiruvella KK, Liang Z, Wilson TE (2013) Repair of double-strand breaks by end joining. Cold Spring Harb Perspect Biol 5:a012757. CrossRefGoogle Scholar
  8. 8.
    Karanam K, Kafri R, Loewer A, Lahav G (2012) Quantitative live cell imaging reveals a gradual shift between DNA repair mechanisms and a maximal use of HR in mid S phase. Mol Cell 47:320–329. CrossRefGoogle Scholar
  9. 9.
    Shim EY et al (2010) Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks. EMBO J 29:3370–3380. CrossRefGoogle Scholar
  10. 10.
    Postow L (2011) Destroying the ring: freeing DNA from Ku with ubiquitin. FEBS Lett 585:2876–2882. CrossRefGoogle Scholar
  11. 11.
    Postow L et al (2008) Ku80 removal from DNA through double strand break-induced ubiquitylation. J Cell Biol 182:467–479. CrossRefGoogle Scholar
  12. 12.
    Ismail IH et al (2015) The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice. Nat Cell Biol 17:1446–1457. CrossRefGoogle Scholar
  13. 13.
    Feng L, Chen J (2012) The E3 ligase RNF8 regulates KU80 removal and NHEJ repair. Nat Struct Mol Biol 19:201–206. CrossRefGoogle Scholar
  14. 14.
    Ishida N et al (2017) Ubiquitylation of Ku80 by RNF126 promotes completion of nonhomologous end joining-mediated DNA repair. Mol Cell Biol 37:e00347-16. CrossRefGoogle Scholar
  15. 15.
    Nishi R et al (2018) The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage. Sci Rep 8:17891. CrossRefGoogle Scholar
  16. 16.
    Abdul Rehman SA et al (2016) MINDY-1 Is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes. Mol Cell 63:146–155. CrossRefGoogle Scholar
  17. 17.
    Nijman SM et al (2005) A genomic and functional inventory of deubiquitinating enzymes. Cell 123:773–786. CrossRefGoogle Scholar
  18. 18.
    Bekker-Jensen S, Mailand N (2015) RNF138 joins the HR team. Nat Cell Biol 17:1375–1377. CrossRefGoogle Scholar
  19. 19.
    Huang OW et al (2012) Phosphorylation-dependent activity of the deubiquitinase DUBA. Nat Struct Mol Biol 19:171–175. CrossRefGoogle Scholar
  20. 20.
    Mevissen TE et al (2013) OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 154:169–184. CrossRefGoogle Scholar
  21. 21.
    Britton S, Coates J, Jackson SP (2013) A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. J Cell Biol 202:579–595. CrossRefGoogle Scholar
  22. 22.
    Pierce AJ, Johnson RD, Thompson LH, Jasin M (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13:2633–2638CrossRefGoogle Scholar
  23. 23.
    Ogiwara H et al (2011) Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors. Oncogene 30:2135–2146. CrossRefGoogle Scholar
  24. 24.
    Chapman JR, Taylor MR, Boulton SJ (2012) Playing the end game: DNA double-strand break repair pathway choice. Mol Cell 47:497–510. CrossRefGoogle Scholar
  25. 25.
    Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271. CrossRefGoogle Scholar
  26. 26.
    Ma HT, Poon RY (2017) Synchronization of HeLa cells. Methods Mol Biol 1524:189–201. CrossRefGoogle Scholar
  27. 27.
    Gupta R et al (2018) DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity. Cell 173:972–988. CrossRefGoogle Scholar
  28. 28.
    Pierce AJ, Hu P, Han M, Ellis N, Jasin M (2001) Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev 15:3237–3242. CrossRefGoogle Scholar
  29. 29.
    Grob P et al (2012) Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV. DNA Repair (Amst) 11:74–81. CrossRefGoogle Scholar
  30. 30.
    Wu D, Topper LM, Wilson TE (2008) Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae. Genetics 178:1237–1249. CrossRefGoogle Scholar
  31. 31.
    Niu H, Raynard S, Sung P (2009) Multiplicity of DNA end resection machineries in chromosome break repair. Genes Dev 23:1481–1486. CrossRefGoogle Scholar
  32. 32.
    Postow L, Funabiki H (2013) An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation. Cell Cycle 12:587–595. CrossRefGoogle Scholar
  33. 33.
    de Vivo A et al (2019) The OTUD5-UBR5 complex regulates FACT-mediated transcription at damaged chromatin. Nucleic Acids Res 47:729–746. CrossRefGoogle Scholar
  34. 34.
    Difilippantonio MJ et al (2000) DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature 404:510–514. CrossRefGoogle Scholar
  35. 35.
    Lim DS et al (2000) Analysis of ku80-mutant mice and cells with deficient levels of p53. Mol Cell Biol 20:3772–3780CrossRefGoogle Scholar
  36. 36.
    Wei S et al (2012) Ku80 functions as a tumor suppressor in hepatocellular carcinoma by inducing S-phase arrest through a p53-dependent pathway. Carcinogenesis 33:538–547. CrossRefGoogle Scholar
  37. 37.
    Shan J, Zhao W, Gu W (2009) Suppression of cancer cell growth by promoting cyclin D1 degradation. Mol Cell 36:469–476. CrossRefGoogle Scholar
  38. 38.
    Yu M et al (2016) USP11 is a negative regulator to gammaH2AX ubiquitylation by RNF8/RNF168. J Biol Chem 291:959–967. CrossRefGoogle Scholar
  39. 39.
    Rodrigue A et al (2006) Interplay between human DNA repair proteins at a unique double-strand break in vivo. EMBO J 25:222–231. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Fangzhou Li
    • 1
  • Qianqian Sun
    • 1
  • Kun Liu
    • 1
  • Haichao Han
    • 1
  • Ning Lin
    • 1
  • Zhongyi Cheng
    • 3
  • Yueming Cai
    • 4
  • Feng Tian
    • 2
  • Zebin Mao
    • 1
  • Tanjun Tong
    • 1
  • Wenhui Zhao
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, Peking University Health Science CenterBeijing Key Laboratory of Protein Posttranslational Modifications and Cell FunctionBeijingChina
  2. 2.Department of Laboratory Animal SciencePeking University Health Science CenterBeijingChina
  3. 3.Jingjie PTM BioLab, Co. Ltd, Hangzhou Economic and Technological Development AreaHangzhouChina
  4. 4.Rheumatic Immunology DepartmentPeking University Shenzhen HospitalShenzhenChina

Personalised recommendations