A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M

Abstract

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...

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…A recent work by Prakash et al (2009) described the ability of purified Mph1 to bind D-loop structures and to unwind these DNA structures, although it is probably not its only function since it would not explain the mph1 mutator phenotype (Scheller et al, 2000). S. cerevisiae Mph1 shows sequence similarity to human FANCM and the archaeabacterial Hef (Komori et al, 2002;Meetei et al, 2005). FANCM belongs to the Fanconi Anemia (FA) pathway (Meetei et al, 2005) regulating a cellular response to genotoxic stresses (Kennedy and D'Andrea, 2005).…”

“…S. cerevisiae Mph1 shows sequence similarity to human FANCM and the archaeabacterial Hef (Komori et al, 2002;Meetei et al, 2005). FANCM belongs to the Fanconi Anemia (FA) pathway (Meetei et al, 2005) regulating a cellular response to genotoxic stresses (Kennedy and D'Andrea, 2005). It has a conserved helicaseATPase domain showing ATP-dependent DNA translocase activity and an endonuclease domain homologous to S. cerevisiae Mus81 endonuclease (Meetei et al, 2005).…”

“…FANCM belongs to the Fanconi Anemia (FA) pathway (Meetei et al, 2005) regulating a cellular response to genotoxic stresses (Kennedy and D'Andrea, 2005). It has a conserved helicaseATPase domain showing ATP-dependent DNA translocase activity and an endonuclease domain homologous to S. cerevisiae Mus81 endonuclease (Meetei et al, 2005). The archaeabacterial Hef protein consists of two domains; the helicase domain similar to Mph1 and the C-terminal domain with some sequence similarity to Mus81 (Komori et al, 2002).…”

Targeting Fanconi Anemia/BRCA2 Pathway Defects in Cancer: The Significance of Preclinical Pharmacogenomic Models

“…1). The formation of the nuclear FA core complex, comprising Fanca, Fancb, Fancc, Fance, Fancf, Fancg, Fancl, Fancm, and one yet unidentified 100-kDa protein (Faap100), depends on the integrity of all proteins involved (11,20,21). The FA complex becomes activated on DNA damage, as coordinated by DNA damage sensor proteins such as ataxia telangiectasia mutated (ATM) or ataxia telangiectasia mutated and Rad3-related (ATR) (13, 22 -25).…”

“…A direct function of the FA pathway in DNA repair is further supported by Fancj being identical to the DNA helicase Brip1 (Brca1-interacting protein; refs. 10,35,36) and Fancm representing the human orthologue of the bacterial DNA repair protein Hef (11,37).…”