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 is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in 1) destabilization of FANCM and MHF2, 2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, 3) defective chromatin localization of FA nuclear core complex proteins, 4) elevated MMC-induced chromosome aberrations, and 5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo III␣ and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo III␣-BLAP75/ RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo III␣ and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.[Keywords: RECQ; double Holliday junction; Chromatin; BLAP18/RMI2; BLM] Supplemental material is available at http://www.genesdev.org.