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The vertebrate Hef ortholog is a component of the Fanconi anemia tumor-suppressor pathway

Nature Structural & Molecular Biology volume 12, pages 763–771 (2005)Cite this article

Abstract

The helicase-associated endonuclease for fork-structured DNA (Hef) is an archaeabacterial protein that processes blocked replication forks. Here we have isolated the vertebrate Hef ortholog and investigated its molecular function. Disruption of this gene in chicken DT40 cells results in genomic instability and sensitivity to DNA cross-links. The similarity of this phenotype to that of cells lacking the Fanconi anemia–related (FA) tumor-suppressor genes led us to investigate whether Hef functions in this pathway. Indeed, we found a genetic interaction between the FANCC and Hef genes. In addition, Hef is a component of the FA nuclear protein complex that facilitates its DNA damage–inducible chromatin localization and the monoubiquitination of the FA protein FANCD2. Notably, Hef interacts directly with DNA structures that are intermediates in DNA replication. This discovery sheds light on the origins, regulation and molecular function of the FA tumor-suppressor pathway in the maintenance of genome stability.

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Figure 1: The N-terminal Mph1-like domain of vertebrate Hef has DNA binding and ATPase activity.
Figure 2: Disruption of the GgHef locus results in reduced survival and chromosome breakage in response to DNA cross-links.
Figure 3: A genetic interaction between Hef and FANCC.
Figure 4: ΔHef cells are defective at homologous recombination repair in response to cross-links but not to double strand breaks.
Figure 5: Hef is a component of the human FA complex and contributes to its E3 ligase activity.
Figure 6: Hef ablation compromises the chicken FA core complex and impairs its accumulation in chromatin.

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References

  1. Lehmann, A.R. DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie 85, 1101–1111 (2003).

    Article  CAS  Google Scholar 

  2. Tian, M., Shinkura, R., Shinkura, N. & Alt, F.W. Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF. Mol. Cell. Biol. 24, 1200–1205 (2004).

    Article  CAS  Google Scholar 

  3. Sargent, R.G. et al. Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells. Nucleic Acids Res. 28, 3771–3778 (2000).

    Article  CAS  Google Scholar 

  4. McPherson, J.P. et al. Involvement of mammalian Mus81 in genome integrity and tumor suppression. Science 304, 1822–1826 (2004).

    Article  CAS  Google Scholar 

  5. Whitby, M.C., Osman, F. & Dixon, J. Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4. J. Biol. Chem. 278, 6928–6935 (2003).

    Article  CAS  Google Scholar 

  6. Komori, K., Fujikane, R., Shinagawa, H. & Ishino, Y. Novel endonuclease in Archaea cleaving DNA with various branched structure. Genes Genet. Syst. 77, 227–241 (2002).

    Article  CAS  Google Scholar 

  7. Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y. & Morikawa, K. Crystal structure and functional implications of Pyrococcus furiosus hef helicase domain involved in branched DNA processing. Structure (Camb) 13, 143–153 (2005).

    Article  CAS  Google Scholar 

  8. Nishino, T., Komori, K., Ishino, Y. & Morikawa, K. X-ray and biochemical anatomy of an archaeal XPF/Rad1/Mus81 family nuclease: similarity between its endonuclease domain and restriction enzymes. Structure (Camb) 11, 445–457 (2003).

    Article  CAS  Google Scholar 

  9. Komori, K. et al. Cooperation of the N-terminal helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks. J. Biol. Chem. 279, 53175–53185 (2004).

    Article  CAS  Google Scholar 

  10. Schurer, K.A., Rudolph, C., Ulrich, H.D. & Kramer, W. Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from homologous recombination, but not from postreplicative repair. Genetics 166, 1673–1686 (2004).

    Article  Google Scholar 

  11. Prakash, R. et al. Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3′ to 5′ DNA helicase. J. Biol. Chem. 280, 7854–7860 (2005).

    Article  CAS  Google Scholar 

  12. Scholz, B., Rechter, S., Drach, J.C., Townsend, L.B. & Bogner, E. Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovirus. Nucleic Acids Res. 31, 1426–1433 (2003).

    Article  CAS  Google Scholar 

  13. Rocak, S., Emery, B., Tanner, N.K. & Linder, P. Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs. Nucleic Acids Res. 33, 999–1009 (2005).

    Article  CAS  Google Scholar 

  14. Sonoda, E., Takata, M., Yamashita, Y.M., Morrison, C. & Takeda, S. Homologous DNA recombination in vertebrate cells. Proc. Natl. Acad. Sci. USA 98, 8388–8394 (2001).

    Article  CAS  Google Scholar 

  15. Simpson, L.J. & Sale, J.E. Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line. EMBO J. 22, 1654–1664 (2003).

    Article  CAS  Google Scholar 

  16. Yamamoto, K. et al. Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination. Mol. Cell. Biol. 25, 34–43 (2005).

    Article  CAS  Google Scholar 

  17. Niedzwiedz, W. et al. The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair. Mol. Cell 15, 607–620 (2004).

    Article  CAS  Google Scholar 

  18. Hatanaka, A. et al. Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination. Mol. Cell. Biol. 25, 1124–1134 (2005).

    Article  CAS  Google Scholar 

  19. Nakanishi, K. et al. Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair. Proc. Natl. Acad. Sci. USA 102, 1110–1115 (2005).

    Article  CAS  Google Scholar 

  20. Pierce, A.J., Johnson, R.D., Thompson, L.H. & Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13, 2633–2638 (1999).

    Article  CAS  Google Scholar 

  21. Arakawa, H., Hauschild, J. & Buerstedde, J.M. Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295, 1301–1306 (2002).

    Article  CAS  Google Scholar 

  22. Garcia-Higuera, I. et al. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell 7, 249–262 (2001).

    Article  CAS  Google Scholar 

  23. Meetei, A.R. et al. A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome. Mol. Cell. Biol. 23, 3417–3426 (2003).

    Article  CAS  Google Scholar 

  24. Qiao, F., Moss, A. & Kupfer, G.M. Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner. J. Biol. Chem. 276, 23391–23396 (2001).

    Article  CAS  Google Scholar 

  25. Mi, J. & Kupfer, G.M. The Fanconi anemia core complex associates with chromatin during S phase. Blood 105, 759–766 (2005).

    Article  CAS  Google Scholar 

  26. D'Andrea, A.D. & Grompe, M. The Fanconi anaemia/BRCA pathway. Nat. Rev. Cancer 3, 23–34 (2003).

    Article  CAS  Google Scholar 

  27. Joenje, H. & Patel, K.J. The emerging genetic and molecular basis of Fanconi anaemia. Nat. Rev. Genet. 2, 446–457 (2001).

    Article  CAS  Google Scholar 

  28. Scheller, J., Schurer, A., Rudolph, C., Hettwer, S. & Kramer, W. MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage. Genetics 155, 1069–1081 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  Google Scholar 

  30. Eddy, S.R. Profile hidden Markov models. Bioinformatics 14, 755–763 (1998).

    Article  CAS  Google Scholar 

  31. Bateman, A. et al. The Pfam protein families database. Nucleic Acids Res. 32, D138–D141 (2004).

    Article  CAS  Google Scholar 

  32. Chenna, R. et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497–3500 (2003).

    Article  CAS  Google Scholar 

  33. Schmidt, H.A., Strimmer, K., Vingron, M. & von Haeseler, A. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, 502–504 (2002).

    Article  CAS  Google Scholar 

  34. Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

    Article  CAS  Google Scholar 

  35. Pace, P. et al. FANCE: the link between Fanconi anaemia complex assembly and activity. EMBO J. 21, 3414–3423 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

G.M. was supported by a grant from Children's Leukaemia Trust, W.N. by a postdoctoral fellowship from Association of International Cancer Research, A.A. by a postdoctoral fellowship from Leukaemia Research Fund and F.L. by a grant from the Fanconi Anaemia Research Fund. We thank H. Joenje and J. DeWinter for the gift of the FANCA antibody and FA cell lines.

Author information

Author notes

  1. Georgina Mosedale, Wojciech Niedzwiedz and Arno Alpi: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Georgina Mosedale, Wojciech Niedzwiedz, Arno Alpi, Franco Perrina, Jose B Pereira-Leal, Mark Johnson, Frederic Langevin, Paul Pace & Ketan J Patel

  2. Department of Medicine, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QH, UK

    Ketan J Patel

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  1. Georgina Mosedale
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Correspondence to Ketan J Patel.

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Supplementary information

Supplementary Fig. 1

Evolutionary relationships in the Mph1-like helicases and XPF-like nucleases. (PDF 503 kb)

Supplementary Fig. 2

Clustal alignments of the vertebrate Hef orthologues. (PDF 1030 kb)

Supplementary Fig. 3

Disruption of the chicken HEF gene. (PDF 1022 kb)

Supplementary Fig. 4

Generation of an in-situ tagged FANCC and FANCF DT40 strain. (PDF 752 kb)

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Mosedale, G., Niedzwiedz, W., Alpi, A. et al. The vertebrate Hef ortholog is a component of the Fanconi anemia tumor-suppressor pathway. Nat Struct Mol Biol 12, 763–771 (2005). https://doi.org/10.1038/nsmb981

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