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RAD18 transmits DNA damage signalling to elicit homologous recombination repair

Nature Cell Biology volume 11, pages 592–603 (2009)Cite this article

Abstract

To maintain genome stability, cells respond to DNA damage by activating signalling pathways that govern cell-cycle checkpoints and initiate DNA repair. Cell-cycle checkpoint controls should connect with DNA repair processes, however, exactly how such coordination occurs in vivo is largely unknown. Here we describe a new role for the E3 ligase RAD18 as the integral component in translating the damage response signal to orchestrate homologous recombination repair (HRR). We show that RAD18 promotes homologous recombination in a manner strictly dependent on its ability to be recruited to sites of DNA breaks and that this recruitment relies on a well-defined DNA damage signalling pathway mediated by another E3 ligase, RNF8. We further demonstrate that RAD18 functions as an adaptor to facilitate homologous recombination through direct interaction with the recombinase RAD51C. Together, our data uncovers RAD18 as a key factor that orchestrates HRR through surveillance of the DNA damage signal.

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Figure 1: RAD18 forms DNA DSB-induced foci.
Figure 2: RNF8/UBC13 is required for DSB-induced RAD18 recruitment.
Figure 3: The ZNF domain of RAD18 is required for RAD18 localization to the sites of DNA damage.
Figure 4: RAD18 promotes homologous recombination.
Figure 5: RNF8 participates in homologous recombination.
Figure 6: RAD18 interacts with RAD51C.
Figure 7: RAD18-binding is critical for RAD51C function in homologous recombination.
Figure 8: RAD18 participates in two distinct DNA repair pathways.

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References

  1. Hoeijmakers, J. H. Genome maintenance mechanisms for preventing cancer. Nature 411, 366–374 (2001).

    Article  CAS  Google Scholar 

  2. Friedberg, E. C. DNA damage and repair. Nature 421, 436–440 (2003).

    Article  Google Scholar 

  3. Bartkova, J. et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870 (2005).

    Article  CAS  Google Scholar 

  4. Gorgoulis, V. G. et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434, 907–913 (2005).

    Article  CAS  Google Scholar 

  5. Bartek, J. & Lukas, J. DNA damage checkpoints: from initiation to recovery or adaptation. Curr. Opin. Cell Biol. 19, 238–245 (2007).

    Article  CAS  Google Scholar 

  6. Fernandez-Capetillo, O., Lee, A., Nussenzweig, M. & Nussenzweig, A. H2AX: the histone guardian of the genome. DNA Repair (Amst.) 3, 959–967 (2004).

    Article  CAS  Google Scholar 

  7. Bassing, C. H. et al. Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors. Cell 114, 359–370 (2003).

    Article  CAS  Google Scholar 

  8. Celeste, A. et al. H2AX haploinsufficiency modifies genomic stability and tumor susceptibility. Cell 114, 371–383 (2003).

    Article  CAS  Google Scholar 

  9. Kim, H., Huang, J. & Chen, J. CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response. Nature Struct. Mol. Biol. 14, 710–715 (2007).

    Article  CAS  Google Scholar 

  10. Huen, M. S. et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 131, 901–914 (2007).

    Article  CAS  Google Scholar 

  11. Wang, B. & Elledge, S. J. Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage. Proc. Natl Acad. Sci. USA 104, 20759–20763 (2007).

    Article  CAS  Google Scholar 

  12. Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 131, 887–900 (2007).

    Article  CAS  Google Scholar 

  13. Kolas, N. K. et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318, 1637–1640 (2007).

    Article  CAS  Google Scholar 

  14. Plans, V. et al. The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation. J. Cell. Biochem 97, 572–582 (2006).

    Article  CAS  Google Scholar 

  15. Weinstock, D. M., Richardson, C. A., Elliott, B. & Jasin, M. Modeling oncogenic translocations: distinct roles for double-strand break repair pathways in translocation formation in mammalian cells. DNA Repair (Amst.) 5, 1065–1074 (2006).

    Article  CAS  Google Scholar 

  16. Lukas, J. & Bartek, J. Watching the DNA repair ensemble dance. Cell 118, 666–668 (2004).

    Article  CAS  Google Scholar 

  17. Kennedy, R. D. & D'Andrea, A. D. DNA repair pathways in clinical practice: lessons from pediatric cancer susceptibility syndromes. J. Clin. Oncol. 24, 3799–3808 (2006).

    Article  CAS  Google Scholar 

  18. West, S. C. Molecular views of recombination proteins and their control. Nature Rev. Mol. Cell Biol. 4, 435–445 (2003).

    Article  CAS  Google Scholar 

  19. Thacker, J. The RAD51 gene family, genetic instability and cancer. Cancer Lett. 219, 125–135 (2005).

    Article  CAS  Google Scholar 

  20. Masson, J. Y. et al. Identification and purification of two distinct complexes containing the five RAD51 paralogs. Genes Dev. 15, 3296–3307 (2001).

    Article  CAS  Google Scholar 

  21. Sigurdsson, S. et al. Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange. Genes Dev. 15, 3308–3318 (2001).

    Article  CAS  Google Scholar 

  22. Tateishi, S., Sakuraba, Y., Masuyama, S., Inoue, H. & Yamaizumi, M. Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. Proc. Natl Acad. Sci. USA 97, 7927–7932 (2000).

    Article  CAS  Google Scholar 

  23. Tateishi, S. et al. Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells. Mol. Cell. Biol. 23, 474–481 (2003).

    Article  CAS  Google Scholar 

  24. Hoege, C., Pfander, B., Moldovan, G. L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    Article  CAS  Google Scholar 

  25. Watanabe, K. et al. Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination. EMBO J. 23, 3886–3896 (2004).

    Article  CAS  Google Scholar 

  26. Szuts, D., Simpson, L. J., Kabani, S., Yamazoe, M. & Sale, J. E. Role for RAD18 in homologous recombination in DT40 cells. Mol. Cell. Biol. 26, 8032–8041 (2006).

    Article  CAS  Google Scholar 

  27. Saberi, A. 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. 27, 2562–2571 (2007).

    Article  CAS  Google Scholar 

  28. Zhao, G. Y. et al. A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol. Cell 25, 663–675 (2007).

    Article  CAS  Google Scholar 

  29. Morris, J. R. & Solomon, E. BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair. Hum. Mol. Genet. 13, 807–817 (2004).

    Article  CAS  Google Scholar 

  30. Polanowska, J., Martin, J. S., Garcia-Muse, T., Petalcorin, M. I. & Boulton, S. J. A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites. EMBO J. 25, 2178–2188 (2006).

    Article  CAS  Google Scholar 

  31. Kim, H., Chen, J. & Yu, X. Ubiquitin-binding protein RAP80 mediates BRCA1-dependent DNA damage response. Science 316, 1202–1205 (2007).

    Article  CAS  Google Scholar 

  32. Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007).

    Article  CAS  Google Scholar 

  33. Wang, B. et al. Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316, 1194–1198 (2007).

    Article  CAS  Google Scholar 

  34. Petrini, J. H. Cell signaling. A touching response to damage. Science 316, 1138–1139 (2007).

    Article  CAS  Google Scholar 

  35. Sartori, A. A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).

    Article  CAS  Google Scholar 

  36. Yamashita, Y. M. et al. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. EMBO J. 21, 5558–5566 (2002).

    Article  CAS  Google Scholar 

  37. Yoshimura, A. et al. A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin. DNA Repair (Amst.) 5, 1307–1316 (2006).

    Article  CAS  Google Scholar 

  38. Weinstock, D. M., Nakanishi, K., Helgadottir, H. R. & Jasin, M. Assaying double-strand break repair pathway choice in mammalian cells using a targeted endonuclease or the RAG recombinase. Methods Enzymol. 409, 524–540 (2006).

    Article  CAS  Google Scholar 

  39. Sorensen, C. S. et al. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nature Cell Biol. 7, 195–201 (2005).

    Article  CAS  Google Scholar 

  40. French, C. A., Tambini, C. E. & Thacker, J. Identification of functional domains in the RAD51L2 (RAD51C) protein and its requirement for gene conversion. J. Biol. Chem. 278, 45445–45450 (2003).

    Article  CAS  Google Scholar 

  41. Kuznetsov, S. et al. RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females. J. Cell Biol. 176, 581–592 (2007).

    Article  CAS  Google Scholar 

  42. Liu, Y., Tarsounas, M., O'Regan, P. & West, S. C. Role of RAD51C and XRCC3 in genetic recombination and DNA repair. J. Biol. Chem. 282, 1973–1979 (2007).

    Article  CAS  Google Scholar 

  43. Reynolds, J. Resolving a Holliday romance. Nature Cell Biol. 6, 184 (2004).

    Article  CAS  Google Scholar 

  44. Liu, Y., Masson, J. Y., Shah, R., O'Regan, P. & West, S. C. RAD51C is required for Holliday junction processing in mammalian cells. Science 303, 243–246 (2004).

    Article  CAS  Google Scholar 

  45. French, C. A. et al. Role of mammalian RAD51L2 (RAD51C) in recombination and genetic stability. J. Biol. Chem. 277, 19322–19330 (2002).

    Article  CAS  Google Scholar 

  46. Godthelp, B. C. et al. Mammalian Rad51C contributes to DNA cross-link resistance, sister chromatid cohesion and genomic stability. Nucleic Acids Res. 30, 2172–2182 (2002).

    Article  CAS  Google Scholar 

  47. Hinz, J. M., Helleday, T. & Meuth, M. Reduced apoptotic response to camptothecin in CHO cells deficient in XRCC3. Carcinogenesis 24, 249–253 (2003).

    Article  CAS  Google Scholar 

  48. Takata, M. et al. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol. Cell. Biol. 21, 2858–2866 (2001).

    Article  CAS  Google Scholar 

  49. Rodrigue, A. et al. Interplay between human DNA repair proteins at a unique double-strand break in vivo. EMBO J. 25, 222–231 (2006).

    Article  CAS  Google Scholar 

  50. Chen, J. et al. Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell 2, 317–328 (1998).

    Article  CAS  Google Scholar 

  51. Maser, R. S., Zinkel, R. & Petrini, J. H. An alternative mode of translation permits production of a variant NBS1 protein from the common Nijmegen breakage syndrome allele. Nature Genet 27, 417–421 (2001).

    Article  CAS  Google Scholar 

  52. Hofer, B., Backhaus, S. & Timmis, K. N. The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes. Gene 144, 9–16 (1994).

    Article  CAS  Google Scholar 

  53. Yu, X., Fu, S., Lai, M., Baer, R. & Chen, J. BRCA1 ubiquitinates its phosphorylation-dependent binding partner CtIP. Genes Dev. 20, 1721–1726 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Shiomi for HCT116 RAD18−/− cells, M. Yamaizumi for RAD18−/− MEFs, S. Akira for UBC13-deficient cells, H.Nagasawa for IRS3 and V79 cells, M. Jasin for U2OS cells with DR–GFP integration and pCBASce plasmids, B. P. Chen for NU7441 and J. Groenendyk for helping with the BIAcore system. J.C would like to thank all colleagues for discussions and technical assistance, and J. Wood for proofreading. This work was supported by grants from the National Institutes of Health to J.C., M.S.Y.H. is supported by the Anna Fuller Fund Fellowship and J.C. is a recipient of an Era of Hope Scholar award from the Department of Defense (a member of the Mayo Clinic Breast SPORE program).

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Author notes

  1. Hongtae Kim

    Present address: Current address: Department of Biological Science, Sungkyunkwan University, 300 Chunchundong, Suwon 440–746, Korea.,

  2. Xiaochun Yu

    Present address: Current address: Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, 109 Zina Pitcher Place, BSRB 1520, Ann Arbor, MI 48109, USA.,

Authors and Affiliations

  1. Department of Therapeutic Radiology, Yale University School of Medicine, P.O. Box 208040, New Haven, 06520, CT, USA

    Jun Huang, Michael S. Y. Huen, Hongtae Kim & Junjie Chen

  2. Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

    Charles Chung Yun Leung & J N Mark Glover

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Contributions

J.H. performed most of the experiments; J.H. and J.C. designed the experiments, analysed the data and wrote the manuscript; M.S.Y.H. performed the experiments shown in Fig. 2e; H.K. performed the experiments shown in Fig. S2a; C.C.Y.L. and J.N.M.G. performed the experiments shown in Fig. S2b, c and X.Y. generated the RNF8 and RAP80 knockout mice.

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Correspondence to Junjie Chen.

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The authors declare no competing financial interests.

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Huang, J., Huen, M., Kim, H. et al. RAD18 transmits DNA damage signalling to elicit homologous recombination repair. Nat Cell Biol 11, 592–603 (2009). https://doi.org/10.1038/ncb1865

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