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...