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• W2041473497 abstract "Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-FboxSkp2 ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex. Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-FboxSkp2 ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex. Fanconi anemia (FA) 3The abbreviations used are: FA, Fanconi anemia; MMC, mitomycin C; TPR, tetratricopeptide repeat; CTD, C-terminal domain; HEAT, Huntington elongation A subunit target of rapamycin; E3, ubiquitin-protein isopeptide ligase; MES, 4-morpholineethanesulfonic acid. is an inherited chromosomal instability disorder manifesting a variety of congenital malformations, pancytopenia, and a predisposition to cancer (1Auerbach A.D. Buchwald M. Joenje H. Begelstein B. Kinzler K.W. Genetics of Human Cancer. McGraw-Hill, New York2002: 317-322Google Scholar, 2Joenje H. Patel K.J. Nat. Rev. Genet. 2001; 2: 446-457Crossref PubMed Scopus (507) Google Scholar). Cells from FA patients are particularly sensitive to DNA cross-linking agents such as mitomycin C (MMC) (3German J. Schonberg S. Caskie S. Warburton D. Falk C. Ray J.H. Blood. 1987; 69: 1637-1641Crossref PubMed Google Scholar) and diepoxybutane (4Auerbach A.D. Blood. 1988; 72: 366-367Crossref PubMed Google Scholar), suggestive of defects in a pathway that normally promotes genomic stability by responding to cross-link damage. FA is a genetically heterogeneous disorder for which at least 12 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, and M) have been identified to date. Genes defective in 11 of the complementation groups have been cloned and shown to cooperate in a common biochemical pathway (5de Winter J.P. Leveille F. van Berkel C.G. Rooimans M.A. van Der Weel L. Steltenpool J. Demuth I. Morgan N.V. Alon N. Bosnoyan-Collins L. Lightfoot J. Leegwater P.A. Waisfisz Q. Komatsu K. Arwert F. Pronk J.C. Mathew C.G. Digweed M. Buchwald M. Joenje H. Am. J. Hum. Genet. 2000; 67: 1306-1308Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 6de Winter J.P. Rooimans M.A. van Der Weel L. van Berkel C.G. Alon N. Bosnoyan-Collins L. de Groot J. Zhi Y. Waisfisz Q. Pronk J.C. Arwert F. Mathew C.G. Scheper R.J. Hoatlin M.E. Buchwald M. Joenje H. Nat. Genet. 2000; 24: 15-16Crossref PubMed Scopus (236) Google Scholar, 7de Winter J.P. Waisfisz Q. Rooimans M.A. van Berkel C.G. Bosnoyan-Collins L. Alon N. Carreau M. Bender O. Demuth I. Schindler D. Pronk J.C. Arwert F. Hoehn H. Digweed M. Buchwald M. Joenje H. Nat. Genet. 1998; 20: 281-283Crossref PubMed Scopus (283) Google Scholar, 8The Fanconi Anemia/Breast Cancer ConsortiumNat. Genet. 1996; 14: 324-328Crossref PubMed Scopus (261) Google Scholar, 9Howlett N.G. Taniguchi T. Olson S. Cox B. Waisfisz Q. De DieSmulders C. Persky N. Grompe M. Joenje H. Pals G. Ikeda H. Fox E.A. D'Andrea A.D. Science. 2002; 297: 606-609Crossref PubMed Scopus (968) Google Scholar, 10Lo Ten Foe J.R. Rooimans M.A. Bosnoyan-Collins L. Alon N. Wijker M. Parker L. Lightfoot J. Carreau M. Callen D.F. Savoia A. Cheng N.C. van Berkel C.G. Strunk M.H. Gille J.J. Pals G. Kruyt F.A. Pronk J.C. Arwert F. Buchwald M. Joenje H. Nat. Genet. 1996; 14: 320-323Crossref PubMed Scopus (302) Google Scholar, 11Meetei A.R. de Winter J.P. Medhurst A.L. Wallisch M. Waisfisz Q. van de Vrugt H.J. Oostra A.B. Yan Z. Ling C. Bishop C.E. Hoatlin M.E. Joenje H. Wang W. Nat. Genet. 2003; 35: 165-170Crossref PubMed Scopus (471) Google Scholar, 12Strathdee C.A. Gavish H. Shannon W.R. Buchwald M. Nature. 1992; 30: 763-767Crossref Scopus (536) Google Scholar, 13Timmers C. Taniguchi T. Hejna J. Reifsteck C. Lucas L. Bruun D. Thayer M. Cox B. Olson S. D'Andrea A.D. Moses R. Grompe M. Mol. Cell. 2001; 7: 241-248Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 14Meetei A.R. Medhurst A.L. Ling C. Xue Y. Singh T.R. Bier P. Steltenpool J. Stone S. Dokal I. Mathew C.G. Hoatlin M. Joenje H. de Winter J.P. Wang W. Nat. Genet. 2005; 37: 958-963Crossref PubMed Scopus (361) Google Scholar). FANCA, B, C, E, F, G, L, and M (11Meetei A.R. de Winter J.P. Medhurst A.L. Wallisch M. Waisfisz Q. van de Vrugt H.J. Oostra A.B. Yan Z. Ling C. Bishop C.E. Hoatlin M.E. Joenje H. Wang W. Nat. Genet. 2003; 35: 165-170Crossref PubMed Scopus (471) Google Scholar, 14Meetei A.R. Medhurst A.L. Ling C. Xue Y. Singh T.R. Bier P. Steltenpool J. Stone S. Dokal I. Mathew C.G. Hoatlin M. Joenje H. de Winter J.P. Wang W. Nat. Genet. 2005; 37: 958-963Crossref PubMed Scopus (361) Google Scholar, 15de Winter J.P. Van der Weel L. de Groot J. Stone S. Waisfisz Q. Arwert F. Scheper R.J. Kruyt F.A. Hoatlin M.E. Joenje H. Hum. Mol. Genet. 2000; 9: 2665-2674Crossref PubMed Scopus (172) Google Scholar, 16Medhurst A.L. Huber P.A. Waisfisz Q. de Winter J.P. Mathew C.G. Hum. Mol. Genet. 2001; 10: 423-429Crossref PubMed Scopus (140) Google Scholar, 17Pace P. Johnson M. Tan W.M. Mosedale G. Sng C. Hoatlin M. de Winter J. Joenje H. Gergely F. Patel K.J. EMBO J. 2002; 21: 3414-3423Crossref PubMed Scopus (138) Google Scholar, 18Waisfisz Q. de Winter J.P. Kruyt F.A. de Groot J. Van der Weel L. Dijkmans L.M. Zhi Y. Arwert F. Scheper R.J. Youssoufian H. Hoatlin M.E. Joenje H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10320-10325Crossref PubMed Scopus (124) Google Scholar) form a putative E3 ubiquitin ligase core complex in the nucleus. The integrity of the core complex is essential for the monoubiquitination of FANCD2 (19Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar) during the S-phase of the cell cycle (20Taniguchi T. Garcia-Higuera I. Andreassen P.R. Gregory R.C. Grompe M. D'Andrea A.D. Blood. 2002; 100: 2414-2420Crossref PubMed Scopus (390) Google Scholar) as well as for the upsurge of FANCD2 monoubiquitination following DNA damage by cross-linking agents, ultraviolet or ionizing radiation, and hydroxyurea-induced nucleotide depletion (19Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, 21Gregory R.C. Taniguchi T. D'Andrea A.D. Semin. Cancer Biol. 2003; 13: 77-82Crossref PubMed Scopus (60) Google Scholar). The recently discovered FA-I complementation group has a defect in FANCD2 monoubiquitination with no apparent effect on the FA core complex (22Levitus M. Rooimans M.A. Steltenpool J. Cool N.F. Oostra A.B. Mathew C.G. Hoatlin M.E. Waisfisz Q. Arwert F. de Winter J.P. Joenje H. Blood. 2004; 103: 2498-2503Crossref PubMed Scopus (198) Google Scholar), suggestive of additional complexity in the FA damage response pathway. Following exposure to DNA-damaging agents, monoubiquitinated FANCD2 moves into the chromatin fraction where it may function in repairing DNA damage by homologous recombination (23Moynahan M.E. Chiu J.W. Koller B.H. Jasin M. Mol. Cell. 1999; 4: 511-518Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, 24Kobayashi J. Antoccia A. Tauchi H. Matsuura S. Komatsu K. DNA Repair (Amst. 2004; 3: 855-861Crossref PubMed Scopus (156) Google Scholar, 25Tutt A. Bertwistle D. Valentine J. Gabriel A. Swift S. Ross G. Griffin C. Thacker J. Ashworth A. EMBO J. 2001; 20: 4704-4716Crossref PubMed Scopus (379) Google Scholar). Only three subunits of the FA core complex include regions of homology to proteins of known function or structure. FANCL possesses an N-terminal WD40 repeat region that mediates interactions with the FA complex (58Gurtan A.M. Stuckert P. D'Andrea A.D. J. Biol. Chem. 2006; 281: 10896-10905Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and a C-terminal plant homeodomain finger domain required for E3 ubiquitin ligase activity (11Meetei A.R. de Winter J.P. Medhurst A.L. Wallisch M. Waisfisz Q. van de Vrugt H.J. Oostra A.B. Yan Z. Ling C. Bishop C.E. Hoatlin M.E. Joenje H. Wang W. Nat. Genet. 2003; 35: 165-170Crossref PubMed Scopus (471) Google Scholar). The human FANCM protein is an ortholog of the archaeabacterial helicase-associated endonuclease for fork-structured DNA and contains both DNA helicase-like and endonuclease-like domains (14Meetei A.R. Medhurst A.L. Ling C. Xue Y. Singh T.R. Bier P. Steltenpool J. Stone S. Dokal I. Mathew C.G. Hoatlin M. Joenje H. de Winter J.P. Wang W. Nat. Genet. 2005; 37: 958-963Crossref PubMed Scopus (361) Google Scholar), suggesting a direct role for the FA core complex in the processing of DNA damage. FANCG contains several putative tetratricopeptide repeats (TPRs) (27Blom E. van de Vrugt H.J. de Vries Y. de Winter J.P. Arwert F. Joenje H. DNA Repair (Amst. 2004; 3: 77-84Crossref PubMed Scopus (59) Google Scholar). TPR domains typically serve as protein interaction modules within multisubunit enzymes (28Blatch G.L. Lassle M. BioEssays. 1999; 21: 932-939Crossref PubMed Scopus (968) Google Scholar) or as scaffolds for the assembly of multiprotein complexes (29D'Andrea L.D. Regan L. Trends Biochem. Sci. 2003; 28: 655-662Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). Previously characterized TPR repeats consist of a 34-residue motif in tandem arrays of 3-16 copies. Although TPR motifs exhibit a considerable degree of sequence diversity, their structures are remarkably similar. The remaining FA core complex proteins have a predicted α-helical character suggestive of helical repeats that might function as a scaffold that interacts with DNA damage response or repair proteins. The FANCF protein is a component of the FA core complex that is necessary for FANCD2 monoubiquitination (15de Winter J.P. Van der Weel L. de Groot J. Stone S. Waisfisz Q. Arwert F. Scheper R.J. Kruyt F.A. Hoatlin M.E. Joenje H. Hum. Mol. Genet. 2000; 9: 2665-2674Crossref PubMed Scopus (172) Google Scholar). Methylation of the FANCF gene promoter results in the abrogation of FANCF function in a subset of ovarian, oral, lung, and cervical cancers (30Taniguchi T. Tischkowitz M. Ameziane N. Hodgson S.V. Mathew C.G. Joenje H. Mok S.C. D'Andrea A.D. Nat. Med. 2003; 9: 568-574Crossref PubMed Scopus (478) Google Scholar, 31Marsit C.J. Liu M. Nelson H.H. Posner M. Suzuki M. Kelsey K.T. Oncogene. 2004; 23: 1000-1004Crossref PubMed Scopus (192) Google Scholar, 32Narayan G. Arias-Pulido H. Nandula S.V. Basso K. Sugirtharaj D.D. Vargas H. Mansukhani M. Villella J. Meyer L. Schneider A. Gissmann L. Durst M. Pothuri B. Murty V.V. Cancer Res. 2004; 64: 2994-2997Crossref PubMed Scopus (169) Google Scholar). The C-terminal domain (CTD) of FANCF interacts with FANCG, as shown by yeast two-hybrid analysis, co-immunoprecipitation, and in vitro studies (15de Winter J.P. Van der Weel L. de Groot J. Stone S. Waisfisz Q. Arwert F. Scheper R.J. Kruyt F.A. Hoatlin M.E. Joenje H. Hum. Mol. Genet. 2000; 9: 2665-2674Crossref PubMed Scopus (172) Google Scholar). The N-terminal region of FANCF engages in interactions with the FANCC:FANCE subcomplex (33Leveille F. Blom E. Medhurst A.L. Bier P. Laghmani E.H. Johnson M. Rooimans M.A. Sobeck A. Waisfisz Q. Arwert F. Patel K.J. Hoatlin M.E. Joenje H. de Winter J.P. J. Biol. Chem. 2004; 279: 39421-39430Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In addition, both N- and C-terminal regions of FANCF synergistically support one another's interactions with their respective partners. For example, mutations in the C-terminal half of FANCF partially compromise interactions with FANCC and with FANCE, proteins that bind to the N-terminal half of FANCF (33Leveille F. Blom E. Medhurst A.L. Bier P. Laghmani E.H. Johnson M. Rooimans M.A. Sobeck A. Waisfisz Q. Arwert F. Patel K.J. Hoatlin M.E. Joenje H. de Winter J.P. J. Biol. Chem. 2004; 279: 39421-39430Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The synergistic interactions of the N- and C-terminal halves of FANCF with the FANCA:FANCG and FANCC:FANCE subcomplexes, respectively, engendered the notion that FANCF functions as a physical adaptor that is essential for the integrity of the FA core nuclear complex (33Leveille F. Blom E. Medhurst A.L. Bier P. Laghmani E.H. Johnson M. Rooimans M.A. Sobeck A. Waisfisz Q. Arwert F. Patel K.J. Hoatlin M.E. Joenje H. de Winter J.P. J. Biol. Chem. 2004; 279: 39421-39430Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Our x-ray crystallographic studies of FANCF reveal that the C-terminal domain of FANCF comprises a series of helical hairpins arranged in a right handed solenoid. Four loops inserted between these α-helical hairpins constitute a significant portion of the protein surface. Residues located in two of these loops are critical for the interaction of FANCF with FANCA: FANCG, a prerequisite for FANCD2 monoubiquitination and normal cellular tolerance of cross-linking agents like mitomycin C. In addition, the FANCF protein is structurally similar to Cand1, a protein regulator of the SCF ubiquitin ligase complex. Our structure and mutational analyses of FANCF provide direct support for the suggestion that the FA protein complex may share structural characteristics with multiprotein ubiquitin ligases. Protein Expression and Purification—The FANCF156-357 protein was expressed as a thioredoxin fusion using a modified pET32a vector where the enterokinase cleavage site has been replaced with a Prescission protease site. BL21(DE3) cells (Novagen) were induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 6 h at 16 °C. The cells were lysed in lysis buffer (100 mm Tris-HCl, pH 8.0, 100 mm KCl, 5 mm β-mercaptoethanol, 5 mm imidazole, 10% glycerol). The cleared cell lysate was passed through a 5-ml HiTrap HP chelating cartridge (Amersham Biosciences). The column was washed with 500 mm KCl and 20 mm imidazole in the lysis buffer. Protein was eluted by on-resin cleavage with Prescission protease (Amersham Biosciences) in cleavage buffer (50 mm Tris-HCl, pH 8.0, 100 mm KCl, 5 mm β-mercaptoethanol, 10% glycerol) and applied to a 5-ml HiTrap Q cation exchange cartridge. The protein was eluted by a 50-1000 mm KCl gradient over 15 column volumes in buffer Q (50 mm Tris-HCl, pH 8.0, 2 mm dithiothreitol, 0.1 mm EDTA, 1% glycerol). In the final step, the protein was applied to an S-200 size exclusion column equilibrated with buffer S (20 mm Tris-HCl, pH 8.0, 50 mm KCl, 2 mm dithiothreitol, 0.1 mm EDTA, 1% glycerol). Crystallization and Structure Determination—Crystals of FANCF156-357 were grown using the hanging drop vapor diffusion method from a 10 mg/ml protein stock. Crystals appeared overnight and grew to maximum size in 3-5 days at 21 °C in well buffer containing 100 mm Bistris, pH 7.0, 200 mm Li2SO4, 20-25% polyethylene glycol 3350, and 10% ethylene glycol. Two related fragments, FANCF156-368 and FANCF156-374, prepared in the same manner, were tested in crystallization, but both of these proteins failed to produce useful crystals. For diffraction experiments, crystals were equilibrated into cryoprotecting buffer (100 mm Nacacodylate, pH 7.0, 200 mm Li2SO4, 25% polyethylene glycol 3350, 20% glycerol) and cooled in liquid N2. A mercury derivative was prepared by soaking crystals overnight at 21 °C in a cryoprotection buffer containing 0.03 mm ethylmercuric phosphate and 0.03 mm β-mercaptoethanol. The crystals belong to the P3121 space group with cell dimensions a = b = 96.4 Å, c = 48.0 Å, α = β = 90°, and γ = 120°. A single anomalous dispersion data set was collected on the X-12C beamline at the National Synchrotron Light Source in Upton, NY. Diffraction data were processed in HKL2000 (34Otwinowski Z. Minor W. Carter C Jr W. Sweet R.M. Macromolecular Crystallography. 276. Academic Press, New York1997: 307-326Google Scholar). The program SOLVE (35Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) was used to locate one mercury site in the asymmetric unit. After solvent flattening, a partial model built using RESOLVE (36Terwilliger T.C. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2002; 58: 1937-1940Crossref PubMed Scopus (283) Google Scholar) was used as a starting point for manual modeling with the program O (37Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The model was refined with REFMAC5 (38Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) using the single anomalous dispersion x-ray data extending to a resolution of 2.4 Å. Cell Lines and Culture Conditions—Epstein-Barr virus-transformed lymphoblasts were maintained in RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum and grown in a humidified atmosphere of 5% CO2 at 37 °C. Wild-type (normal adult) lymphoblast lines (39Taniguchi T. D'Andrea A.D. Blood. 2002; 100: 2457-2462Crossref PubMed Scopus (66) Google Scholar) (PD7 and GM02554) and the FA lymphoblast line EUFA121 (FA-F) were used in this study. Mammalian Expression Vectors—The retroviral expression vector pMMP-puro was described previously (40Ory D.S. Neugeboren B.A. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11400-11406Crossref PubMed Scopus (801) Google Scholar). pMMP-puro-FLAG-FANCF was generated by adding the FLAG tag (DYKDDDDK) to the N terminus of FANCF. Production of pMMP retroviral supernatants was performed as previously described (41Naf D. Kupfer G.M. Suliman A. Lambert K. D'Andrea A.D. Mol. Cell. Biol. 1998; 18: 5952-5960Crossref PubMed Scopus (107) Google Scholar). FANCF mutations were generated by site-directed PCR mutagenesis (QuikChange Kit, Stratagene). Truncations and internal deletions of FANCF were generated with standard PCR cloning techniques. Retroviral Infection—FA-F lymphoblasts underwent three rounds of infection with supernatant from 293GPG fibroblasts transfected with pMMP-FANCF. Cells were exposed to the virus-containing supernatant for 24 h in the presence of 8 μg/ml polybrene (Sigma) followed by incubation for 24 h in regular RPMI (15% fetal bovine serum). After the final round, the infected cells were washed free of viral supernatant and resuspended in growth medium. After 48-72 h, the cells were transferred to medium containing 1 μg/ml puromycin. Dead cells were removed over Ficoll-Paque Plus (Amersham Biosciences AB, Uppsala, Sweden) cushion after 5 days, and the remaining cells were grown under continuous selection in puromycin. MMC Sensitivity and Chromosomal Breakage Assays—The MMC sensitivity of transduced lymphoblasts was determined as described previously (42Yamashita T. Barber D.L. Zhu Y. Wu N. D'Andrea A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6712-6716Crossref PubMed Scopus (126) Google Scholar) but using the CyQuant cell proliferation assay kit (Invitrogen). Chromosome breakage analysis was performed by the Cytogenetics Core Facility of the Dana-Farber Cancer Institute, as described previously (43Andreassen P.R. D'Andrea A.D. Taniguchi T. Genes Dev. 2004; 18: 1958-1963Crossref PubMed Scopus (336) Google Scholar). Antibodies—Antibodies against FANCA (N-terminal) (44Kupfer G.M. Naf D. Suliman A. Pulsipher M. D'Andrea A.D. Nat. Genet. 1997; 17: 487-490Crossref PubMed Scopus (159) Google Scholar), FANCD2 (E35) (19Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar), FANCF (45Siddique M.A. Nakanishi K. Taniguchi T. Grompe M. D'Andrea A.D. Exp. Hematol. 2001; 29: 1448-1455Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and FANCG (46Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell. Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar) have been described previously. Anti-FLAG antibody (monoclonal M5 or rabbit polyclonal) was purchased from Sigma Aldrich. Immunoprecipitations—Whole cell extracts were prepared in lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% (v/v) Triton X-100) supplemented with protease tablets (Roche Applied Science). Each lysate was normalized to contain 2 mg of total protein, and FLAG-FANCF was immunoprecipitated with 50 μl of packed M2 anti-FLAG-agarose (Sigma Aldrich) for 24 h at 4 °C. The agarose was then washed 3-4 times with chilled lysis buffer. FLAG-FANCF was eluted with either 150 ng/ml FLAG peptide or with SDS sample buffer (Bio-Rad) containing 5% β-mercaptoethanol. Immunoblotting—Cells were lysed with 2× Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol, boiled for 5 min, and subjected to SDS-PAGE in a 3-8% gradient gel (Invitrogen) for FANCD2 or a 4-12% gradient gel (Invitrogen) with MES buffer (Invitrogen) for FANCA, -F, and -G. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes using a submerged transfer apparatus (Bio-Rad) filled with transfer buffer (Invitrogen). After blocking with 5% nonfat dried milk in TBS-T (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Tween 20), the membrane was incubated with the primary antibody diluted in TBS-T (1:500 dilution for FANCA, -D2, -F, -G; 1:1000 for M5; and 1:250 for rabbit anti-FLAG), washed extensively, and incubated with the appropriate horseradish peroxidase-linked secondary antibody (Amersham Biosciences). Chemiluminescence was used for detection. Generation of DNA Damage—Cells growing in RPMI 1640 medium (Invitrogen) supplemented with 15% fetal bovine serum and l-glutamine were exposed to 2 mm hydroxyurea (Sigma) for 24 h. For mitomycin C (Sigma) treatment, see the MMC sensitivity and chromosomal breakage assays section. The Highly Conserved CTD of FANCF Is a Helical Repeat Motif—Two regions of high sequence conservation are evident when comparing FANCF proteins from human, mouse, toad (Xenopus), and pufferfish, one region near the N terminus, and another close to the C terminus (supplemental Fig. 1). Of special note is the highly conserved C-terminal region of FANCF (Fig. 1, b and c). Eleven of the 23 residues in the Leu-329-Leu-352 stretch are strictly conserved in all species compared, and four additional residues retain a conserved hydrophobic character (e.g. Phe or Tyr at position 339; Ile or Val at position 346). Overall, 43 of 374 residues, or 11.5% in human FANCF, are strictly conserved in orthologous proteins, whereas 48% are identical in the Leu-329-Leu-352 region (Fig. 1b), and the homology reaches 65% if the four conserved hydrophobic residues are included. We therefore focused our attention on the C-terminal region of the FANCF protein, creating several expression constructs for human FANCF fragments based on the aligned sequences. When fused to thioredoxin, the C-terminal region of FANCF was soluble when expressed in Escherichia coli, in contrast to full-length FANCF, which was completely insoluble. Limited proteolysis of this fragment of FANCF further demarcated a domain that was resistant to proteolytic cleavage with trypsin and proteinase K. Both enzymes produced similar, stable fragments of FANCF that were identified by mass spectrometry and N-terminal sequencing (data not shown). On the basis of these results, three additional expression constructs were generated (see “Experimental Procedures”), and these C-terminal fragments were expressed and purified for crystallographic studies. Well diffracting crystals were obtained for the fragment encompassing residues 156-357, whereas fragments with additional C-terminal residues failed to produce useful crystals. The structure of the FANCF156-357 was determined by single anomalous dispersion using crystals derivatized with ethylmercuric phosphate. The crystallographic model was refined against x-ray data extending to 2.4 Å resolution, resulting in a crystallographic R value of 19% and an Rfree value of 25% (Table 1). The structure encompasses residues Leu-329-Leu-352, and it highlights several features of the protein that were targeted for mutagenesis and found to be functionally important.TABLE 1Data collection and refinement statisticsData collectionSpace groupP3121Cell dimensionsa, b, c (Å)96.4, 96.4, 48.0α, β, γ (°)90, 90, 120Resolution (Å)50-2.4 (2.49-2.40)aParentheses refer to the highest resolution shell.Rsym (%)3.8 (26.2)I/σI9.8 (6.2)Completeness (%)99.6 (97.0)Redundancy7.1 (6.8)RefinementResolution range24.0-2.4No. reflections9,109Rwork/Rfree0.19/0.25No. atomsProtein1,344Water80B-factors (Å2)52.5Root mean square deviationsBond lengths (Å)0.024Bond angles (°)2.421a Parentheses refer to the highest resolution shell. Open table in a new tab FANCF Has Structural Similarity to Cand1—We searched the Protein Data Bank for proteins that were structurally homologous to FANCF using the DALI (47Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3566) Google Scholar) server. The CTD of FANCF is most structurally similar to the Cand1 regulatory subunit of the SCF ubiquitin ligase complex (48Goldenberg S.J. Cascio T.C. Shumway S.D. Garbutt K.C. Liu J. Xiong Y. Zheng N. Cell. 2004; 119: 517-528Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) (Z score = 7.0), the Cse1p exportin of the Cse1p:Kap60p:RanGTP nuclear export complex (49Matsuura Y. Stewart M. Nature. 2004; 432: 872-877Crossref PubMed Scopus (164) Google Scholar) (Z score = 5.9), and a hypothetical Epsin N-terminal homology-Vps 27/Hrs/STAM domain AT3G16270 from Arabidopsis thaliana (Z score = 5.8) (59Lopez-Mendez B. Pantoja-Uceda D. Tomizawa T. Koshiba S. Kigawa T. Shirouzu M. Terada T. Inoue M. Yabuki T. Aoki M. Seki E. Matsuda T. Hirota H. Yoshida M. Tanaka A. Osanai T. Seki M. Shinozaki K. Yokoyama S. Guntert P. J. Biomol. NMR. 2006; 29: 205-206Crossref Scopus (18) Google Scholar). The Cand1 protein forms an extended solenoid that coils around the SCF scaffold Cul1 (48Goldenberg S.J. Cascio T.C. Shumway S.D. Garbutt K.C. Liu J. Xiong Y. Zheng N. Cell. 2004; 119: 517-528Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Both the Cand1 and Cse1p proteins are composed of Huntington elongation A subunit target of rapamycin (HEAT) repeats. The HEAT repeats are 40-residue-long, two-helix motifs often found in tandem arrays within functionally divergent proteins (50Andrade M.A. Petosa C. O'Donoghue S.I. Muller C.W. Bork P. J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (400) Google Scholar). The core region of FANCF encompassing helices α1-α6 of FANCF CTD also bears superficial resemblance to the armadillo helical repeats of importin-α (51Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar) and related proteins. However, the candidate armadillo repeats of FANCF do not have hydrophobic core residues in the canonical positions of a true armadillo repeat, indicative of a different packing arrangement between α-helices in the FANCF CTD. Surface Loop Residues Are Critical for Proper FANCF Function—The C-terminal domain of FANCF (CTD, residues 156-357) consists almost entirely of α-helices connected by several loops, with overall dimensions of 58 × 38 × 29 Å (Fig. 1a). Successive pairs of antiparallel helices, or helical hairpins, are arranged in a right-handed solenoid with an extended shape. The hairpins are composed of helices ranging in lengths from 9 to 18 residues. Four hairpin repeats (HP1-HP4) are formed by helices α2-α9 and capped on either end by helices α1 and α10 (Fig. 1a). Short connecting loops join the helices within each hairpin, and the longer connections joining the hairpins together consist of loops that vary in length from 4 to 23 residues. The N-terminal helix (helix α1) of the CTD fragment is amphipathic with an outer, solvent-accessible surface decorated with charged and hydrophilic residues Ser-162 and Thr-165 and Glu-168, -172, and -176. This feature suggests that the CTD fragment derived by proteolytic digestion of FANCF encompasses a complete domain of helical hairpin repeats and that helix α1 is solvent-exposed in full-length FANCF. The" @default.
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• W2041473497 title "Structural Determinants of Human FANCF Protein That Function in the Assembly of a DNA Damage Signaling Complex" @default.
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