Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

Fanconi anemia (FA) is a genetic disease featuring genomic instability and cancer predisposition 1 . Nine FA genes have been identified, and their products participate in a DNA damage response network involving BRCA1 and BRCA2 2,3 . We have previously purified a FA core complex containing the FANCL ubiquitin ligase and 6 other FA proteins 4-6 . Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the FA DNA damage response pathway 2,7 . Here we show that another component of this complex, FAAP250, is mutated in FA patients of a new complementation group (FA-M). FAAP250, renamed FANCM, has sequence similarity to known DNA repair proteins, including archaeal Hef, yeast Mph1 and human ERCC4/ XPF. FANCM can dissociate DNA triplex, possibly due to its ability to translocate on duplex DNA. FANCM is essential for FANCD2 monoubiquitination and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between FA proteins and DNA repair; FANCM may act as an engine that translocates the FA core complex along DNA. KeywordsFanconi anemia; FANCM; Hef; MPH1; XPF/ERCC4; FANCD2 #: Correspondence should be addressed to JPW and WW. Telephone: 410-558-8334 (WW); 31-020-444-8283 (JPW), Fax: 410-558-8331 (WW); 31-020-444-8285 (JPW), Email:E-mail: wangw@grc.nia.nih.gov (WW);E-mail: j.dewinter@vumc.nl (JPW). Competing Interests StatementThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2009 July 1. Published in final edited form as:Nat Genet. 2005 September ; 37(9): 958-963. doi:10.1038/ng1626. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe have previously shown that 7 out of 9 components of the FA core complex are FA proteins (FANC-A, B, C, E, F, G, and L) 4-6 . Using mass spectrometry, we identified another component, FAAP250 (Fig. 1a), as KIAA1596, a hypothetical protein with unknown function. Antibodies raised against KIAA1596 specifically recognized the 250 kD polypeptide of the FA core complex immunopurified by a FANCA antibody, supporting the identity of KIAA1596 as FAAP250 (Fig. 1b).Several lines of evidence suggest that FAAP250 is an integral component of the FA core complex. First, FAAP250 was detected in the FA core complex immunoisolated by an antiFlag antibody from cells expressing either Flag-tagged FANCA, or Flag-tagged FANCL (Fig. 1b). Second, FAAP250 was coimmunoprecipitated by antibodies against multiple FA core components components (FANCA, C, and F) from lymphoblastoid cells of a normal individual, but not from patient cells deficient in the corresponding FA proteins (Fig. 1c). Third, reciprocal immunoprecipitation in HeLa cells using the FAAP250 antibody showed co-precipitation of multiple FA core complex components, such as FANCL, FANCA, and FANCG ( Fig. 1d and data not shown).Importantly, depletion of FAAP250 in HeLa and HEK293 cells by siRNA drastically reduced the levels of monoubiquitinated FANCD2 u...

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.

SUMMARY FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here we show that FANCM forms a conserved DNA remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA remodeling complex that protects replication forks from yeast to human.

The Fanconi anemia (FA) core complex plays a crucial role in a DNA damage response network with BRCA1 and BRCA2. How this complex interacts with damaged DNA is unknown, as only the FA core protein FANCM (the homolog of an archaeal helicase/nuclease known as HEF) exhibits DNA binding activity. Here, we describe the identification of FAAP24, a protein that targets FANCM to structures that mimic intermediates formed during the replication/repair of damaged DNA. FAAP24 shares homology with the XPF family of flap/fork endonucleases, associates with the C-terminal region of FANCM, and is a component of the FA core complex. FAAP24 is required for normal levels of FANCD2 monoubiquitylation following DNA damage. Depletion of FAAP24 by siRNA results in cellular hypersensitivity to DNA crosslinking agents and chromosomal instability. Our data indicate that the FANCM/FAAP24 complex may play a key role in recruitment of the FA core complex to damaged DNA.

The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.

Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.

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.