Fanconi anemia (FA) is a recessively inherited disease characterized at the cellular level by spontaneous chromosomal instability and specific hypersensitivity to cross-linking agents. FA is genetically heterogeneous, comprising at least eight complementation groups (A-H). We report that the protein encoded by the gene mutated in complementation group G (FANCG) localizes to the cytoplasm and nucleus of the cell and assembles in a molecular complex with the FANCA protein, both in vivo and in vitro. Endogenous FANCA͞FANCG complex was detected in both non-FA cells and in FA cells from groups D and E. By contrast, no complex was detected in specific cell lines belonging to groups A and G, whereas reduced levels were found in cells from groups B, C, F, and H. Wild-type levels of FANCA͞ FANCG complex were restored upon correction of the cellular phenotype by transfection or cell fusion experiments, suggesting that this complex is of functional significance in the FA pathway. These results indicate that the cellular FA phenotype can be connected to three biochemical subtypes based on the levels of FANCA͞FANCG complex. Disruption of the complex may provide an experimental strategy for chemosensitization of neoplastic cells.

Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur "upstream" in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2. IntroductionMultiple pathways are required by the cell to deal with endogenous and exogenous damage to the DNA and to maintain the genome's integrity. The inactivation of these pathways leads to an unstable genome, which increases the risk of tumorigenesis. Many of the genes involved in DNA repair and genomic stability are mutated in cancer predisposition syndromes such as XPA-G (xeroderma pigmentosum), NBS1 (Nijmegen breakage syndrome), and ATM (ataxia telangiectasia). The function of the gene products that are defective in the Fanconi anemia (FA) pathway, which is speculated to act in DNA repair, remains elusive.FA is clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy, especially acute myeloid leukemia (AML) and squamous cell carcinoma (SCC). Cells derived from patients with FA are hypersensitive to DNA cross-linking agents, such as mitomycin C (MMC) and diexpoxybutane (DEB), which suggests that the FA pathway may be involved in the sensing and/or repair of interstrand cross-links. Cell fusion experiments have identified 12 different complementation groups, and 11 of their corresponding disease genes have been cloned : FANCA, FANCB, FANCC, FANCD1/ BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCJ/BRIP1, FANCL, Many of the FA proteins interact in a nuclear complex, the assembly of which is required for the monoubiquitination of a downstream target, FANCD2. 15 This protein has been the subject of intense investigation in recent years, and several studies have revealed that the monoubiquitinated isoform of FANCD2 enters discrete nuclear foci where it colocalizes with multiple proteins involved in genomic stability including BRCA1, NBS1, and RAD51. [15][16][17] Furthermore, it has b...

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.

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