Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair

Koji Nakanishi

Yun Yang

Andrew Pierce

Toshiyasu Taniguchi

^{Molecular Biology Program, Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, New York, NY 10021;}^{International Agency for Research on Cancer, 150 Cours Albert Thomas, 69008 Lyon, France;}^{Department of Radiation Oncology, Dana–Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115; and}^{Institut für Humangenetik, Charite-Campus Virchow-Klinikum, Humboldt Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany}

^{To whom correspondence should be addressed. E-mail:}gro.ccksm.iks@nisaj-m.

^{Present address: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536.}

^{Present address: Fred Hutchinson Cancer Research Center, Seattle, WA 98109.}

Edited by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, and approved December 2, 2004

Received 2004 Oct 20

Abstract

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca^{cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.}

Keywords: double-strand break repair, FANC, homologous recombination, mammalian cells

Abstract

Cellular DNA repair defects in a number of different pathways are associated with tumor susceptibility and developmental defects in humans and mice. Recent work has specifically implicated defects in homologous DNA repair in tumor predisposition in the hereditary breast cancer syndromes (1, 2). Mechanistically, pathways that use sequence homology for DNA repair are broadly characterized into two types based on whether homologous associations arise from DNA strand exchange or strand annealing (3). Homologous recombination, also termed homology-directed repair (HDR), utilizes strand exchange in a gene conversion reaction involving a single-strand and a DNA duplex, and is a major repair pathway in mammalian cells for DNA damage such as double-strand breaks (DSBs) (4).

The other DSB repair pathway using sequence homology is single-strand annealing (SSA), which involves the annealing of complementary single strands formed after resection at a DSB. The biological relevance of this pathway is uncertain, but it is a highly efficient mechanism of DSB repair in mammalian cells involving direct repeats (4). Because a large portion of the mammalian genome consists of repeat sequences, SSA could potentially be an important alternative pathway of homologous repair.

Although HDR and SSA involve a common intermediate (single strands formed after end resection), the subsequent strand exchange and strand-annealing steps, respectively, involve some distinct components. Proteins critical for HDR in mammalian cells include the strand exchange protein RAD51 (5, 6) and the products of the hereditary breast cancer susceptibility genes BRCA1 (7, 8) and BRCA2 (9, 10). BRCA2 directly interacts with RAD51, possibly to promote the strand invasion step of HDR (2, 11). Proteins involved in SSA in mammalian cells include RAD52 (6), which promotes strand annealing in vitro (12). Recent observations suggest a competitive interaction between HDR and SSA in mammalian cells: when either RAD51 or BRCA2 is disrupted, HDR is decreased and SSA is enhanced (6). Such a competitive interaction has also been reported in yeast for several HDR genes (13). However, some proteins may act in both pathways. For example, when BRCA1 function is disrupted, both HDR and SSA are decreased (6). This finding has led to the proposal that BRCA1 may have a role early in homologous repair before the branch point of the HDR and SSA pathways (6).

Cells from patients with Fanconi anemia (FA) have defects in DNA repair, because they are sensitive to DNA-damaging agents and exhibit chromosome aberrations (14, 15). The FANC proteins, which are disrupted in FA patients, have been implicated in HDR, although several reports are contradictory. Impaired DNA damage-induced RAD51 focus formation, which is often associated with HDR defects, has been reported in one study to be a characteristic of cells from the FA-D1 group (i.e., BRCA2) but not of cells from other FA complementation groups (16), whereas another study has reported attenuated RAD51 focus formation in cells from several FA groups (17). In apparent contradiction to these studies, patient fibroblasts representing several upstream FA groups have been reported to have highly elevated levels of homologous recombination between plasmids, suggesting that the FANC proteins suppress HDR (18). By contrast, however, studies in a highly recombinogenic chicken DT40 cell line have implicated the FANC proteins in promoting HDR, such that fancg or fancc mutant cells have either a severe or mild impairment of HDR, respectively (19, 20). Surprisingly, the fancc mutant chicken cells have an increased level of another indicator of recombination, that of sister chromatid exchange (20), which is not observed in cells from human FA complementation groups. To clarify the role of the FANC proteins in homologous repair mammalian cells, we have examined DSB repair in patient-derived FA cells from three complementation groups, as well as mouse cells with a targeted mutation, and report on these results.

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Acknowledgments

We thank members of the M.J. laboratory, especially Nicole Christ, Mingguang Han, Jeremy Stark, Hiroshi Saeki, and Mary Ellen Moynahan, for technical help and intellectual contributions. This work was supported by the Fanconi Anemia Research Fund, the Julie Laub Fund, the Hecksher Foundation, the Fritz-Thyssen-Stiftung (to M.D.), National Institutes of Health Grant P01 CA94060, and National Institutes of Health Grant GM54668 (to M.J.).

Acknowledgments

Notes

Author contributions: K.N., A.J.P., M.D., Z.-Q.W., and M.J. designed research; K.N., Y.-G.Y., and A.J.P. performed research; K.N., Y.-G.Y., A.J.P., T.T., M.D., and A.D.D. contributed new reagents/analytic tools; K.N., Y.-G.Y., and Z.-Q.W. analyzed data; and K.N. and M.J. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: FA, Fanconi anemia; DSB, double-strand break; HDR, homology-directed repair/homologous recombination; SSA, single-strand annealing; NHEJ, nonhomologous end-joining.

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: FA, Fanconi anemia; DSB, double-strand break; HDR, homology-directed repair/homologous recombination; SSA, single-strand annealing; NHEJ, nonhomologous end-joining.

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