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Fanconi anemia (FA) is a rare, heritable chromosomal instability disease characterized by several congenital defects and cancer predisposition. Functional interactions between specific FA proteins and DNA damage response and repair activities have been reported, but the interplay between these mechanisms for maintaining genomic stability are not well understood. Many DNA damage response proteins are transcriptionally regulated by the tumor suppressor protein p53 (TP53), suggesting an important regulatory role for the DNA damage and stress response pathway. To better understand the association between FA and the DNA damage stress response we analyzed the levels of chromosomal damage and damage mediated gene transcription responses in lymphoblastoid cells derived from normal individuals and patients carrying the most common FA complementation group (FA-A). Chromosomal aberrations were first measured after exposure to mitomicyn C (MMC) or hydroxyurea (HU). Aliquots of the same cell were than assayed for the transcriptional response of 21 DNA damage and stress response genes using quantitative real-time PCR. The FA-A lymphoblastoid cells showed significant increases in the frequency of chromosome aberrations relative to non-FA-A lymphoblastoid lines after MMC treatment. The MMC induced damage was correlated with a general increase in expression of TP53-modulated DNA damage stress response genes involved in processes such as DNA repair, cell cycle progression, and apoptosis. Following HU treatment FA cells showed a decreased induction of CAs with much less transcriptional differences between targeted genes. Overall, the differences between the normal and FA-A cells after genotoxic treatments imply an increased activation and reliance of FA cells on the down-stream activities of TP53 for prevention of cell killing and chromosome damage from interstrand crosslinks but not for general replication arrest and double strand breaks. Furthermore, these results imply a regulatory connection between the FA pathway and activation of TP53 for responding to DNA damage. Alterations in the regulation of the DNA damage response may be related to the complex phenotypes seen in FA patients.

The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are largely unknown. By constructing and characterizing a null fancg mutant (KO40) of hamster CHO cells, we show that FancG protects cells against a broad spectrum of genotoxic agents. KO40 is consistently hypersensitive to both alkylating agents that produce monoadducts and those that produce interstrand crosslinks. KO40 cells were no more sensitive to mitomycin C (3x) and diepoxybutane (2x) than to 6-thioguanine (5x), ethylnitrosourea (3x), or methyl methanesulfonate (MMS) (3x). These results contrast with the pattern of selective sensitivity to DNA crosslinking agents seen historically with cell lines from FA patients. The hypersensitivity of KO40 to MMS was not associated with a higher level of initial DNA single-strand breaks; nor was there a defect in removing MNU-induced methyl groups from DNA. Both control and MMS-treated synchronized G1-phase KO40 cells progressed through S phase at a normal rate but showed a lengthening of G2 phase compared with wild type. MMS-treated and untreated early S-phase KO40 cells had increased levels of Rad51 foci compared with wild type. Asynchronous KO40 treated with ionizing radiation (IR) exhibited a normal Rad51 focus response, consistent with KO40 having only slight sensitivity to killing by IR. The plating efficiency and doubling time of KO40 cells were nearly normal, and they showed no increase in spontaneous chromosomal aberrations or sister chromatid exchanges. Collectively, our results do not support a role for FancG during DNA replication that deals specifically with processing DNA crosslinks. Nor do they suggest that the main function of the FA protein "pathway" is to promote efficient homologous recombination. We propose that the primary function of FA proteins is to maintain chromosomal continuity by stabilizing replication forks that encounter nicks, gaps, or replication-blocking lesions.

Abstract: Fanconi anemia (FA) is a rare cancer predisposition disease caused by mutations in at least 12 genes encoding proteins that cooperate to maintain genomic integrity. Variants of FA genes, including FANCG, have been identified in human population screening, but their potential reduction in protein function and role in cancer susceptibility is unclear. To test for possible dysfunction, we constructed plasmids containing four FANCG polymorphisms found in the human population and introduced them in the Fancg-deficient (fancg) KO40 line derived from AA8 hamster CHO cells. Expression of wild-type human FANCG provided fancg cells with complete phenotypic correction as assessed by resistance to the DNA crosslinking agent mitomycin C (MMC), thus providing a sensitive test for detecting the degree of complementation activity for the FANCG variants. We found that all four variants conferred levels of mitomycin C resistance as well as restoration of monoubiquitination of Fancd2, a key indicator of a functional FA protein pathway, similar to those observed in wild-type transfectants. Under the same conditions, the L71P amino acid substitution mutant, identified in an FA patient, gave no complementation. Using this novel system for determining FANCG functionality, we detect no decrement in function of the human FANCG polymorphic variants examined. [Copyright &y& Elsevier]