ATR couples FANCD2 monoubiquitination to the DNA-damage response
Abstract
Fanconi anemia (FA) is a multigenic autosomal recessive cancer susceptibility syndrome. The FA pathway regulates the monoubiquitination of FANCD2 and the assembly of damage-associated FANCD2 nuclear foci. How FANCD2 monoubiquitination is coupled to the DNA-damage response has remained undetermined. Here, we demonstrate that the ATR checkpoint kinase and RPA1 are required for efficient FANCD2 monoubiquitination. Deficiency of ATR function, either in Seckel syndrome, which clinically resembles Fanconi anemia, or by siRNA silencing, results in the formation of radial chromosomes in response to the DNA cross-linker, mitomycin C (MMC), thus mimicking the chromosome instability of FA cells.
Fanconi anemia (FA) is an autosomal recessive disease associated with cancer susceptibility (D'Andrea and Grompe 2003). FA cells are hypersensitive to DNA cross-linking agents, such as mitomycin C (MMC) and cisplatin, and display chromosome instability characterized by the formation of radial chromosomes (D'Andrea and Grompe 2003). These cellular phenotypes indicate that FA cells have a defect in the DNA-damage response.
The genes for eight FA subtypes (A, C, BRCA2/D1, D2, E, F, G, and L) have been cloned, and the encoded FA proteins cooperate in a common pathway. Six of the FA proteins (A, C, E, F, G, and L) assemble into a nuclear complex that is required for the monoubiquitination (activation) of FANCD2 (Garcia-Higuera et al. 2001; D'Andrea and Grompe 2003; Meetei et al. 2003a). Monoubiquitinated FANCD2 is subsequently targeted into DNA repair foci containing BRCA1 (Garcia-Higuera et al. 2001), BRCA2/FANCD1 (Wang et al. 2004), Rad51 (Taniguchi et al. 2002a), and NBS1 (Nakanishi et al. 2002). Deficiencies of FANCD2 monoubiquitination and foci formation, resulting either from mutations in the genes for upstream FA proteins or nonubiquitinable mutants of FANCD2, are associated with cellular hypersensitivity to cross-linking agents and with chromosome instability (Garcia-Higuera et al. 2001).
FANCD2 is monoubiquitinated both during S phase (Taniguchi et al. 2002a) and in response to various DNA damaging agents, including ionizing radiation (IR) and MMC (Garcia-Higuera et al. 2001). The monoubiquitination of FANCD2 is required for its localization to DNA-damage foci (Garcia-Higuera et al. 2001). Although it is clear from these observations that FANCD2 monoubiquitination is activated by DNA damage, it has not been determined how this process is coupled to the DNA-damage response or to S-phase progression.
At the center of the DNA-damage response, the checkpoint protein kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) phosphorylate numerous proteins involved in checkpoint function and DNA repair and thereby coordinate these processes (Abraham 2001; Osborn et al. 2002). It is generally believed that ATM and ATR act in parallel pathways that respond to different DNA stresses, including double-strand breaks (DSBs) and collapse of replication forks (treatment with IR or hydroxyurea [HU], respectively). Whereas ATM phosphorylates FANCD2 in response to IR, we previously demonstrated that ATM-deficient cells are proficient for FANCD2 monoubiquitination and foci formation (Taniguchi et al. 2002b). Thus, ATM is not required for activation of FANCD2 in the FA pathway.
ATR is mutated in one complementation group of Seckel syndrome (SCKL1; O'Driscoll et al. 2003), an autosomal recessive disorder resembling FA, Nijmegen Breakage syndrome, and other diseases involving impaired DNA-damage responses. ATR is present at the replication fork (Lupardus et al. 2002; Tercero et al. 2003). It is activated when the replication fork encounters DNA damage and then phosphorylates several substrates, including its partner kinase, Chk1 (Liu et al. 2000; Zhao and Piwnica-Worms 2001). The RPA complex recruits ATR to newly synthesized, single-strand DNA (ssDNA), generated at sites of damage (Zou and Elledge 2003).
In the present study, we demonstrate using RNA silencing and Seckel syndrome cells that ATR is required for efficient FANCD2 monoubiquitination and FANCD2 foci assembly in response to various genotoxic stresses, including IR and MMC. Further, ATR-deficient Seckel syndrome cells form radial chromosomes in response to MMC, thus establishing a genetic relationship between Seckel syndrome and Fanconi anemia. FANCD2 monoubiquitination also requires the DNA-damage sensor RPA1. This is the first demonstration of a DNA-damage response in which ATR is required for the response to either replication stress or IR without any role for ATM.
Acknowledgments
We thank P. Jeggo and M. O'Driscoll for the primary Seckel fibroblasts, F02-98. The pBABE-hTERTneo plasmid and human ATR cDNA were generously provided by D. Weaver and K. Cimprich, respectively. We thank L. Moreau for chromosome breakage analysis. This work was supported by NIH grants RO1HL52725, RO1DK43889, and PO1HL54785 (A.D.D.). R.P.A. is a Special Fellow of the Leukemia and Lymphoma Society (LLS). T.T. is a Scholar Fellow of the American Society of Hematology.
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Notes
Supplemental material is available at http://www.genesdev.org.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1196104.