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.

  • Original Article
  • Published:

Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end joining

Oncogene volume 34, pages 4403–4411 (2015)Cite this article

Subjects

Abstract

The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18−/−, RNF8−/−, RAD18−/−/RNF8−/− and UBC13−/−clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18−/−, RNF8−/− and RAD18−/−/RNF8−/− clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13−/−cells, they are more sensitive to camptothecin and olaparib than UBC13−/−cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18−/− and RNF8−/− cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation. In contrast, following exposure to γ-rays, RAD18−/−, RNF8−/−, RAD18−/−/RNF8−/− and UBC13−/−cells showed close correlation between cellular survival and Rad51 focus formation at DSB sites. In summary, the current study reveals that Rad18 and Rnf8 facilitate HR by two distinct mechanisms: suppression of the toxic effect of NHEJ on HR during DNA replication and the promotion of Rad51 focus formation at radiotherapy-induced DSB sites.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Yamashita YM, Okada T, Matsusaka T, Sonoda E, Zhao GY, Araki K et al. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. EMBO J 2002; 21: 5558–5566.

    Article  CAS  Google Scholar 

  2. Yoshimura A, Nishino K, Takezawa J, Tada S, Kobayashi T, Sonoda E et al. A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin. DNA Repair (Amst) 2006; 5: 1307–1316.

    Article  CAS  Google Scholar 

  3. Saberi A, Hochegger H, Szuts D, Lan L, Yasui A, Sale JE et al. RAD18 and poly(ADP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair. Mol Cell Biol 2007; 27: 2562–2571.

    Article  CAS  Google Scholar 

  4. Takata M, Sasaki MS, Sonoda E, Morrison C, Hashimoto M, Utsumi H et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 1998; 17: 5497–5508.

    Article  CAS  Google Scholar 

  5. Pommier Y . Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev 2006; 6: 789–802.

    Article  CAS  Google Scholar 

  6. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 2005; 434: 913–917.

    Article  CAS  Google Scholar 

  7. Adachi N, So S, Koyama H . Loss of nonhomologous end joining confers camptothecin resistance in DT40 cells. Implications for the repair of topoisomerase I-mediated DNA damage. J Biol Chem 2004; 279: 37343–37348.

    Article  CAS  Google Scholar 

  8. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005; 434: 917–921.

    Article  CAS  Google Scholar 

  9. Hochegger H, Dejsuphong D, Fukushima T, Morrison C, Sonoda E, Schreiber V et al. Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells. EMBO J 2006; 25: 1305–1314.

    Article  CAS  Google Scholar 

  10. Bunting SF, Callen E, Wong N, Chen HT, Polato F, Gunn A et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 2010; 141: 243–254.

    Article  CAS  Google Scholar 

  11. Li L, Halaby MJ, Hakem A, Cardoso R, El Ghamrasni S, Harding S et al. Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer. J Exp Med 2010; 207: 983–997.

    Article  CAS  Google Scholar 

  12. Santos MA, Huen MS, Jankovic M, Chen HT, Lopez-Contreras AJ, Klein IA et al. Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8. J Exp Med 2010; 207: 973–981.

    Article  CAS  Google Scholar 

  13. Lu LY, Wu J, Ye L, Gavrilina GB, Saunders TL, Yu X . RNF8-dependent histone modifications regulate nucleosome removal during spermatogenesis. Dev Cell 2010; 18: 371–384.

    Article  CAS  Google Scholar 

  14. Sun J, Yomogida K, Sakao S, Yamamoto H, Yoshida K, Watanabe K et al. Rad18 is required for long-term maintenance of spermatogenesis in mouse testes. Mech Dev 2009; 126: 173–183.

    Article  CAS  Google Scholar 

  15. Wu J, Chen Y, Lu LY, Wu Y, Paulsen MT, Ljungman M et al. Chfr and RNF8 synergistically regulate ATM activation. Nat Struct Mol Biol 2011; 18: 761–768.

    Article  CAS  Google Scholar 

  16. Zhao GY, Sonoda E, Barber LJ, Oka H, Murakawa Y, Yamada K et al. A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol Cell 2007; 25: 663–675.

    Article  CAS  Google Scholar 

  17. Yamazoe M, Sonoda E, Hochegger H, Takeda S . Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. DNA Repair (Amst) 2004; 3: 1175–1185.

    Article  CAS  Google Scholar 

  18. Yamamoto M, Okamoto T, Takeda K, Sato S, Sanjo H, Uematsu S et al. Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling. Nat Immunol 2006; 7: 962–970.

    Article  CAS  Google Scholar 

  19. Fukushima T, Matsuzawa S, Kress CL, Bruey JM, Krajewska M, Lefebvre S et al. Ubiquitin-conjugating enzyme Ubc13 is a critical component of TNF receptor-associated factor (TRAF)-mediated inflammatory responses. Proc Natl Acad Sci USA 2007; 104: 6371–6376.

    Article  CAS  Google Scholar 

  20. Adachi N, Suzuki H, Iiizumi S, Koyama H . Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage. J Biol Chem 2003; 278: 35897–35902.

    Article  CAS  Google Scholar 

  21. Maede Y, Shimizu H, Fukushima T, Kogame T, Nakamura T, Miki T et al. Differential and common DNA repair pathways for topoisomerase I- and II-targeted drugs in a genetic DT40 repair cell screen panel. Mol Cancer Ther 2014; 13: 214–220.

    Article  CAS  Google Scholar 

  22. Oestergaard VH, Pentzold C, Pedersen RT, Iosif S, Alpi A, Bekker-Jensen S et al. RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells. DNA Repair (Amst) 2012; 11: 892–905.

    Article  CAS  Google Scholar 

  23. Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 2007; 131: 887–900.

    Article  CAS  Google Scholar 

  24. Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 2007; 318: 1637–1640.

    Article  CAS  Google Scholar 

  25. Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 2007; 131: 901–914.

    Article  CAS  Google Scholar 

  26. Huang J, Huen MS, Kim H, Leung CC, Glover JN, Yu X et al. RAD18 transmits DNA damage signalling to elicit homologous recombination repair. Nat Cell Biol 2009; 11: 592–603.

    Article  CAS  Google Scholar 

  27. Luijsterburg MS, Acs K, Ackermann L, Wiegant WW, Bekker-Jensen S, Larsen DH et al. A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure. EMBO J 2012; 31: 2511–2527.

    Article  CAS  Google Scholar 

  28. Takata M, Sasaki MS, Tachiiri S, Fukushima T, Sonoda E, Schild D et al. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol Cell Biol 2001; 21: 2858–2866.

    Article  CAS  Google Scholar 

  29. Lu CS, Truong LN, Aslanian A, Shi LZ, Li Y, Hwang PY et al. The RING finger protein RNF8 ubiquitinates Nbs1 to promote DNA double-strand break repair by homologous recombination. J Biol Chem 2012; 287: 43984–43994.

    Article  CAS  Google Scholar 

  30. Inagaki A, van Cappellen WA, van der Laan R, Houtsmuller AB, Hoeijmakers JH, Grootegoed JA et al. Dynamic localization of human RAD18 during the cell cycle and a functional connection with DNA double-strand break repair. DNA Repair (Amst) 2009; 8: 190–201.

    Article  CAS  Google Scholar 

  31. Chapman JR, Taylor MR, Boulton SJ . Playing the end game: DNA double-strand break repair pathway choice. Mol Cell 2012; 47: 497–510.

    Article  CAS  Google Scholar 

  32. Watanabe K, Iwabuchi K, Sun J, Tsuji Y, Tani T, Tokunaga K et al. RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1. Nucleic Acids Res 2009; 37: 2176–2193.

    Article  CAS  Google Scholar 

  33. Palle K, Vaziri C . Rad18 E3 ubiquitin ligase activity mediates Fan coni anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition. Cell Cycle 2011; 10: 1625–1638.

    Article  CAS  Google Scholar 

  34. Sonoda E, Sasaki MS, Buerstedde JM, Bezzubova O, Shinohara A, Ogawa H et al. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J 1998; 17: 598–608.

    Article  CAS  Google Scholar 

  35. Ji K, Kogame T, Choi K, Wang X, Lee J, Taniguchi Y et al. A novel approach using DNA-repair-deficient chicken DT40 cell lines for screening and characterizing the genotoxicity of environmental contaminants. Environ Health Perspect 2009; 117: 1737–1744.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the grants-in-aid program of the Ministry of Education, Sports and Culture of Japan (for ST). We thank Drs Tadahiro Shiomi and Naoko Shiomi (National Institute of Radiological Sciences, Japan) for providing us with the HCT116 RAD18−/− and RAD18+/+ cells and Professor Hitoshi Kurumizaka (Waseda University, Japan) for providing us with the anti-Rad51 antibody. Financial support was provided in part by the Uehara Memorial Foundation, the Naito Foundation, Sumitomo Foundation and Kurata Fundation (for KH).

Author information

Author notes

  1. S Nakada

    Present address: 5Current address: Department of Bioregulation and Cellular Response, Graduate School of Medicine, Osaka University, Osaka, Japan.,

Authors and Affiliations

  1. Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto, Japan,

    S Kobayashi, Y Kasaishi, S Era, A Motegi, S Takeda & K Hirota

  2. Center of Integrated Medical Research, School of Medicine, Keio University, Tokyo, Japan

    S Nakada

  3. Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo, Japan

    T Takagi & K Hirota

  4. Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands,

    R K Chiu

Authors
  1. S Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y Kasaishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Nakada
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T Takagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S Era
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A Motegi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R K Chiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S Takeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. K Hirota
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to S Takeda or K Hirota.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kobayashi, S., Kasaishi, Y., Nakada, S. et al. Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end joining. Oncogene 34, 4403–4411 (2015). https://doi.org/10.1038/onc.2014.371

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2014.371

This article is cited by

Search

Advanced search

Quick links