Abstract
In response to DNA damage or replication fork stress, the Fanconi anemia pathway is activated, leading to monoubiquitination of FANCD2 and FANCI and their colocalization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in six conserved and clustered Ser/Thr-Gln motifs of FANCI largely abrogate monoubiquitination and focus formation of both FANCI and FANCD2, resulting in loss of DNA repair function. Conversely, FANCI carrying phosphomimic mutations on the same six residues induces constitutive monoubiquitination and focus formation of FANCI and FANCD2, and protects against cell killing and chromosome breakage by DNA interstrand cross-linking agents. We propose that the multiple phosphorylation of FANCI serves as a molecular switch in activation of the Fanconi anemia pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.
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Acknowledgements
We would like to thank T. Natsume (National Institute of Advanced Industrial Science and Technology, Japan), K. Komatsu (Kyoto University), H. Kurumizaka (Waseda University), J. Lin and J.W. Harper (Harvard Medical School) for reagents; R. Sasaki (Digital Microsystems) for advice and help in microscopy; K. Namikoshi for expert technical help; and H. Shimamoto and S. Arai for secretarial assistance. This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (M.T.), by grants from Ministry of Health, Labour, and Welfare (M.T.), and by US National Institute of Allergy and Infectious Diseases grant 1U19A1067751 and US National Institutes of Health grants (S.J.E.). A. Smogorzewska is supported by T32CA09216 to the MGH Pathology Department. Financial support was also provided by The Novartis Foundation (Japan) for the Promotion of Science (M.T.) and The Uehara Memorial Foundation (M.T.). S.J.E. is funded by the Howard Hughes Medical Institute.
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M.T., A. Smogorzewska and S.J.E. conceived and designed the experiments; M.I., H.K., A. Saberi and E.U. performed most of the DT40 cell experiments; M.I., A.K. and S.T. performed microscopic analysis; E.K., E.K.-K., T.K. and J.T. designed and performed Phos-tag experiments; A. Smogorzewska performed human cell experiments; M.I. and H.K. analyzed the DT40 cell data; M.T., A. Smogorzewska and S.J.E. wrote the paper.
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Ishiai, M., Kitao, H., Smogorzewska, A. et al. FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway. Nat Struct Mol Biol 15, 1138–1146 (2008). https://doi.org/10.1038/nsmb.1504
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DOI: https://doi.org/10.1038/nsmb.1504
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