Search

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by gammaH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

Fanconi anemia (FA) is a multigenic recessive disease resulting in bone marrow failure and increased cancer susceptibility. Cells from FA patients and mouse models are sensitive to DNA interstrand crosslinks (ICLs) and FA mice are moderately sensitive to ionizing radiation (IR). Both kinds of damage induce DNA double strand breaks (DSBs). To date, nine genes in 11 complementation groups have been identified; however, the precise function of the FA pathway remains unclear. Many of the proteins form a nuclear complex necessary for the mono-ubiquitination of the downstream protein, Fancd2. To further investigate the role of the FA pathway in repair of DSBs, we generated Fancd2(-/-)/Prkdc(sc/sc) double mutant mice. Prkdc(sc/sc) mutant mice have a defect in non-homologous end joining (NHEJ) and are sensitive to IR-induced DNA damage. Double mutant animals and primary cells were more sensitive to IR than either single mutant, suggesting that Fancd2 operates in DSB repair pathway distinct from NHEJ. Fancd2(-/-)/Prkdc(sc/sc) double mutant cells were also more sensitive to DSBs generated by a restriction endonuclease. The role of Fancd2 in DSB repair may account for the moderate sensitivity of FA cells to irradiation and FA cells sensitivity to ICLs that are repaired via a DSB intermediate.