P.Pace and M.Johnson contributed equally to this workThe Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identi®ed FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.

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.The hereditary syndrome Fanconi anemia (FA) belongs to a group of caretaker gene diseases characterized by genomic instability and increased susceptibility to cancer. A hallmark of FA is cellular hypersensitivity to DNA interstrand cross-links (ICLs), suggesting a defect in the DNA damage response (18,19,48). Twelve FA complementation groups have been identified, and the majority of the corresponding genes have been cloned (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCJ, FANCL, and FANCM) (22,23,25,43,55,59,60,66,87,93). Although the function of the FA proteins is unknown, identification of BRCA2 (breast cancer-associated gene 2) as FANCD1 and of FANCJ as the BRCA1-associated helicase gene Brip1/BACH1, suggests convergence of the FA/BRCA pathway with a larger network of proteins involved in DNA repair (7,(51)(52)(53)95). This is underscored by the discovery that FANCM is related to archaeal Hef, a protein that binds and processes irregular arrangements of DNA in branched structures resembling replication forks (50,71,72).According to current models, the FA pathway consists of an upstream nuclear core complex, including FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, required for the activation of its target, FANCD2 (24, 34-36, 59, 60, 66). FANCD2 is monoubiquitinated during S phase and in response to various types of DNA damage, including DNA ICLs, DNA double-strand breaks (DSBs), and replication fork stalling (36, 89). DNA damage-induced monoubiquitination of FANCD2 is also reduced in cells from Seckel syndrome patients with a defect in the ataxia telangiectasia-and RAD3-related gene, ATR (1), suggesting that the FA pathway is under at least partial control of the ATR kinase. Monoubiquitination of FANCD2 is required for its association with c...

HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analysed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines, and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression, and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

No abstract