Fanconi anemia (FA) is a human genetic disease characterized by a DNA repair defect and progressive bone marrow failure. Central events in the FA pathway are the monoubiquitination of the Fancd2 protein and the removal of ubiquitin by the deubiquitinating enzyme, Usp1. Here, we have investigated the role of Fancd2 and Usp1 in the maintenance and function of murine hematopoietic stem cells (HSCs). Bone marrow from Fancd22/2 mice and Usp12/2 mice exhibited marked hematopoietic defects. A decreased frequency of the HSC populations including Lin-Sca-11Kit1 cells and cells enriched for dormant HSCs expressing signaling lymphocyte activation molecule (SLAM) markers, was observed in the bone marrow of Fancd2-deficient mice. In addition, bone marrow from Fancd22/2 mice contained significantly reduced frequencies of late-developing cobblestone area-forming cell activity in vitro compared to the bone marrow from wild-type mice. Furthermore, Fancd2-deficient and Usp1-deficient bone marrow had defective long-term in vivo repopulating ability. Collectively, our data reveal novel functions of Fancd2 and Usp1 in maintaining the bone marrow HSC compartment and suggest that FA pathway disruption may account for bone marrow failure in FA patients.

The Fanconi anemia (FA) pathway maintains genomic stability in replicating cells. Some sporadic breast, ovarian, pancreatic, and hematological tumors are deficient in FA pathway function, resulting in sensitivity to DNA-damaging agents. FA pathway dysfunction in these tumors may result in hyperdependence on alternative DNA repair pathways that could be targeted as a treatment strategy. We used a high-throughput siRNA screening approach that identified ataxia telangiectasia mutated (ATM) as a critical kinase for FA pathway-deficient human fibroblasts. Human fibroblasts and murine embryonic fibroblasts deficient for the FA pathway were observed to have constitutive ATM activation and Fancg -/-Atm -/-mice were found to be nonviable. Abrogation of ATM function in FA pathway-deficient cells resulted in DNA breakage, cell cycle arrest, and apoptotic cell death. Moreover, Fanconi anemia complementation group G-(FANCG-) and FANCC-deficient pancreatic tumor lines were more sensitive to the ATM inhibitor KU-55933 than isogenic corrected lines. These data suggest that ATM and FA genes function in parallel and compensatory roles to maintain genomic integrity and cell viability. Pharmaceutical inhibition of ATM may have a role in the treatment of FA pathway-deficient human cancers.

The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCEdeficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.Fanconi anemia (FA) is an inherited cancer susceptibility disorder resulting from germ line disruption of 1 of 11 FA genes (10). The FA proteins cooperate in a common cellular pathway, the FA/BRCA pathway, and disruption of this pathway results in chromosome instability and mitomycin C (MMC) hypersensitivity. Eight of the FA proteins (A, B, C, E, F, G, L, and M) are assembled in a multifunctional FA core complex (complex 1) (22). The complex contains a translocase activity, encoded by the FANCM gene, which is required for its interaction with chromatin (20, 21). The complex also has a monoubiquitin E3 ligase activity, encoded by the FANCL gene, (19) and interacts with a novel E2 ubiquitin (Ub) conjugating enzyme (14). The putative substrate of the Ub ligase is the FANCD2 protein. Monoubiquitinated FANCD2 functions in a downstream complex (complex 2) containing FANCD1/ BRCA2 (11,29). The role of FANCD2-Ub in cross-link repair remains unknown.FANCD2 monoubiquitination occurs during normal S-phase progression (28) or in response to DNA damage (5). ATR-defective (Seckel) cells (1) are defective in the DNA damage-inducible monoubiquitination of FANCD2 and FANCD2 nuclear foci assembly. ATR activates the checkpoint kinase, Chk1 (13,31,32), suggesting that Chk1 may play a role in the FA/BRCA pathway. Consistent with this model, disruption of Chk1 by small interfering RNA (siRNA) or by small-molecule inhibitors (12, 15) results in DNA cross-linker hypersensitivity and chromosome instability, a phenotype reminiscent of FA cells (2). In this paper, we address the role of Chk1 in the regulation of the FA/BRCA pathway. MATERIALS AND METHODSCell culture. HeLa cells, U2OS cells, GM0637 cells, and HEK293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 15% heatinactivated fetal calf serum in a humidified 5% CO 2 incubator at 37°C. DF1179 (FA-E) fibroblasts derived from an FA-E patient were cultured in Chang medium (Irvine Scientific) (generously ...

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81 ؊/؊ , Ercc1 ؊/؊ , and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.The XPF-ERCC1 heterodimer is a structure-specific endonuclease that incises double-strand DNA immediately adjacent to a 3Ј-single-stranded region, removing 3Ј overhangs or opening bubbles (12, 69). ERCC1 is required for DNA binding (74), and XPF harbors the catalytic domain (17). XPF-ERCC1 makes the incision 5Ј to the lesion during nucleotide excision repair (NER), the pathway responsible for removing helixdistorting DNA lesions (69). Defects in NER cause xeroderma pigmentosum (XP), a syndrome characterized by photosensitivity and a dramatically increased risk of skin cancers due to failure to repair UV photolesions. Cells from all XP complementation groups (XP-A to XP-G) and the recently reported ERCC1-deficient patient (33) are hypersensitive to UV irradiation. However, cells deficient in XPF-ERCC1 differ from other XP cells in that they also are exquisitely sensitive to chemicals that induce DNA interstrand cross-links (ICLs) (13,28,54). ICLs are extremely cytotoxic lesions formed when bifunctional agents covalently link both strands of DNA, preventing strand separation, which is necessary for replication or transcription (46). Cross-linking agents such as nitrogen mustards (HN2) (37) and mitomycin C (MMC) (31) produce a mixture of monoadducts and ICLs. However, cytotoxicity correlates with the number of ICLs formed rather than monoadducts (60, 62).ICLs present a unique challenge to cells, in that they affect both strands of DNA and therefore cannot be repaired by a simple excision and resynthesis mechanism. The mechanism of ICL repair in Escherichia coli is well characteri...