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...

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.

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...

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.

The Fanconi anemia (FA) pathway proteins are thought to be involved in the repair of irregular DNA structures including those encountered by the moving replication fork. However, the nature of the DNA structures that recruit and activate the FA proteins is not known. Because FA proteins function within an extended network of proteins, some of which are still unknown, we recently established cell-free assays in Xenopus laevis egg extracts to deconstruct the FA pathway in a fully replication-competent context. Here we show that the central FA pathway protein, xFANCD2, is monoubiquitinated (xFANCD2-L) rapidly in the presence of linear and branched double-stranded DNA (dsDNA) structures but not single-stranded or Yshaped DNA. xFANCD2-L associates with dsDNA structures in an FA core complex-dependent manner but independently of xATRIP, the regulatory subunit of xATR. Formation of xFANCD2-L is also triggered in response to circular dsDNA, suggesting that dsDNA ends are not required to trigger monoubiquitination of FANCD2. The induction of xFANCD2-L in response to circular dsDNA is replication and checkpoint independent. Our results provide new evidence that the FA pathway discriminates among DNA structures and demonstrate that triggering the FA pathway can be uncoupled from DNA replication and ATRIP-dependent activation.Fanconi anemia (FA) belongs to the group of chromosomal instability syndromes including ataxia telangiectasia, Bloom syndrome, and hereditary breast cancer. FA cells are hypersensitive towards DNA interstrand cross-link (ICL)-inducing agents and have highly elevated spontaneous chromosomal breakage rates, suggesting a role for FA proteins in the DNA damage response. The FA pathway consists of an upstream FA "core complex" containing at least eight proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) that is required for the DNA damage-induced activation (monoubiquitination) of its downstream target, FANCD2 (9,12,13,18,27,37). Accumulating evidence supports a role for the FA pathway in DNA doublestrand break (DSB) repair via homologous recombination (HR). Indeed, FA cells are defective in the repair of ICLinduced DNA DSBs mediated by HR (44). Activated FANCD2 interacts with major components of HR repair, such as BRCA1 (breast cancer-associated protein 1), Rad51, and the downstream FA protein BRCA2/FANCD1 (8,12,17,22,47,49); moreover, the BRCA2 interactor PALB2 was recently identified as a Fanconi protein, FANCN (42,43,50). In addition, involvement of the FA proteins in DNA DSB repair via single-strand annealing and nonhomologous end joining has been suggested (10,11,26,36). The recently identified FANCM and FANCJ proteins indicate a direct involvement of FA proteins at sites of DNA repair: FANCM is an FA core complex member (30,31,34) homologous to the archaeal protein Hef (helicase-associated endonuclease for fork-structured DNA), which resolves stalled replication forks (20). FANCJ/BRIP1, which is thought to play a role downstream in the FA pathway (23,24,39), is a 5Ј-to-3Ј DNA helicase with substrate spe...