Fanconi Anemia FANCG Protein in Mitigating Radiation- and Enzyme-Induced DNA Double-Strand Breaks by Homologous Recombination in Vertebrate Cells

Abstract

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased ∼9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.

ACKNOWLEDGMENTS

We thank Akira Shinohara (Osaka University) for the anti-Rad51 antibody; Maria Jasin (Sloan-Kettering Institute) for the SCneo substrate and I-SceI expression vector; Masayo Kimura, Keiko Oka, and Mayu Fujii for expert technical assistance; Hiroko Asahara for help with laser confocal microscopy; Y. Imajo and J. Kubota (Department of Therapeutic Radiology, Kawasaki Medical School) for irradiating cells with the linear accelerator; and Kazuko Hikasa and members of the Central Secretarial Office of Kawasaki Medical School for secretarial assistance. We also thank Shunichi Takeda (Kyoto University) and Ashok Venkitaraman (Cambridge University) for critically reading the manuscript.

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (to M.T. and N.M.) and by grants from the Novartis Foundation, Kobayashi Magobei Foundation, and Yamanouchi Foundation for Research on Metabolic Disorders. Financial support also came from Kawasaki Medical School as project research grants (projects 13-211 and 14-203). A portion of this work was prepared under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract W-7405-ENG-48 and research funded by the Low Dose Radiation Research Program, Biological and Environmental Research (BER), U.S. Department of Energy.