SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype.

DNA interstrand crosslink repair requires several classes of proteins, including structure-specific endonucleases and Fanconi anemia proteins. SLX4, which coordinates three separate endonucleases, was recently recognized as an important regulator of DNA repair. Here we report the first human individuals found to have biallelic mutations in SLX4. These individuals, who were previously diagnosed as having Fanconi anemia, add SLX4 as an essential component to the FA-BRCA genome maintenance pathway.

Fancm-deficient mice reveal unique features of Fanconi anemia complementation group M.

The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.

Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M.

FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM(-/-) cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM(-/-) cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM(-/-) cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex-dependent and -independent manner.

Identification of the Fanconi anemia complementation group I gene, FANCI.

To identify the gene underlying Fanconi anemia (FA) complementation group I we studied informative FA-I families by a genome-wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue-dependent expression. We found a candidate, KIAA1794 on chromosome 15q25-26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA-I individuals. Knock-down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient-derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592.

Evidence for subcomplexes in the Fanconi anemia pathway.

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.

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.

Heterogeneity in Fanconi anemia: evidence for 2 new genetic subtypes.

Fanconi anemia (FA) is an autosomal recessive syndrome featuring diverse symptoms including progressive bone marrow failure and early occurrence of acute myeloid leukemia. Nine genetic subtypes have been described for FA (A, B, C, D1, D2, E, F, G, and L), all of which have been connected to distinct disease genes, except B. Here we report on 8 unrelated FA patients who were excluded from the known subtypes on the basis of phenotypic correction or genetic data. Four of these cell lines failed to complement each other in somatic cell hybrids and therefore represent a new group, termed FA-I. The remaining cell lines complemented group FA-I but did not complement each other, thus representing a second new group, FA-J. Both FA-I and -J cell lines were capable of forming an FA multiprotein core complex. This complex is required for activation of the FANCD2 protein by mono-ubiquitination, a key downstream event in the FA pathway. In FA-I cells FANCD2 was not mono-ubiquitinated, indicating a defect upstream in the FA pathway, whereas in FA-J cells FANCD2 was mono-ubiquitinated, indicating a downstream defect. Our results suggest that the FA pathway of genome stabilization may be controlled by at least 11 different genes, including FANCI and FANCJ.