The nuclear accumulation of the Fanconi anemia protein FANCE depends on FANCC.

The Fanconi anemia (FA) protein FANCE is an essential component of the nuclear FA core complex, which is required for monoubiquitination of the downstream target FANCD2, an important step in the FA pathway of DNA cross-link repair. FANCE is predominantly localized in the nucleus and acts as a molecular bridge between the FA core complex and FANCD2, through direct binding of both FANCC and FANCD2. At present, it is poorly understood how the nuclear accumulation of FANCE is regulated and therefore we investigated the nuclear localization of this FA protein. We found that FANCE has a strong tendency to localize in the nucleus, since the addition of a nuclear export signal does not interfere with the nuclear localization of FANCE. We also demonstrate that the nuclear accumulation of FANCE does not rely solely on its nuclear localization signal motifs, but also on FANCC. The other FA proteins are not involved in the nuclear accumulation of FANCE, indicating a tight relationship between FANCC and FANCE, as suggested from their direct interaction. Finally, we show that the region of FANCE interacting with FANCC appears to be different from the region involved in binding FANCD2. This strengthens the idea that FANCE recruits FANCD2 to the core complex, without interfering with the binding of FANCC.

A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M.

Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.

X-linked inheritance of Fanconi anemia complementation group B.

Fanconi anemia is an autosomal recessive syndrome characterized by diverse clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal instability and susceptibility to cancer. Fanconi anemia has at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in 8 of these have been identified. The gene BRCA2 was suggested to underlie complementation group B, but the evidence is inconclusive. Here we show that the protein defective in individuals with Fanconi anemia belonging to complementation group B is an essential component of the nuclear protein 'core complex' responsible for monoubiquitination of FANCD2, a key event in the DNA-damage response pathway associated with Fanconi anemia and BRCA. Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and subject to X-chromosome inactivation. X-linked inheritance has important consequences for genetic counseling of families with Fanconi anemia belonging to complementation group B. Its presence as a single active copy and essentiality for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable component of the cellular machinery that maintains genomic integrity.

The Fanconi anemia gene product FANCF is a flexible adaptor protein.

The Fanconi anemia (FA) protein FANCF is an essential component of a nuclear core complex that protects the genome against chromosomal instability, but the specific function of FANCF is still poorly understood. Based upon the homology between human and Xenopus laevis FANCF, we carried out an extensive mutagenesis study to examine which domains are functionally important and to gain more insight into the function of FANCF. In contrast to previous suggestions, we show that FANCF does not have a ROM-like function. We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex. The N terminus appears to stabilize the interaction with FANCA and FANCG and is essential for the binding of the FANCC/FANCE subcomplex. We identified several important amino acids in this N-terminal region but, surprisingly, many amino acid changes failed to affect the function of the FANCF protein. Our data demonstrate that FANCF acts as a flexible adaptor protein that plays a key role in the proper assembly of the FA core complex.

FANCD2 protein is expressed in proliferating cells of human tissues that are cancer-prone in Fanconi anaemia.

Fanconi anaemia (FA) is an inherited form of progressive pancytopenia associated with developmental defects, chromosomal instability, and cancer predisposition. At least seven distinct FA proteins function in concert to protect the genome, a key step being the activation of FANCD2 by mono-ubiquitination. This paper reports an immunohistochemical analysis of FANCD2 expression in normal human tissue. The highest expression was observed in maturing spermatocytes and fetal oocytes (consistent with a role for FANCD2 in meiosis) and in germinal centre cells of the spleen, tonsil, and lymph nodes (consistent with a role in proliferation). FANCD2 expression was also seen in tissues predisposed to cancer development in FA patients: haematopoietic cells, especially in the fetus, and squamous cell epithelia, particularly in the head and neck region and uterine cervix. FANCD2 expression was also occasionally seen in the breast and Fallopian tube epithelium, the respiratory epithelium of the trachea, and the exocrine cells of the pancreas, indicating that these tissues may also be cancer-prone in FA. FANCD2 expression is frequently expressed in proliferating cells as demonstrated by Ki-67 immunofluorescence double staining, consistent with a function of FANCD2 in DNA replication.