title: Several tetratricopeptide repeat (TPR) motifs of FANCG are required for assembly of the BRCA2/D1-D2-G-X3 complex, FANCD2 monoubiquitylation and phleomycin resistance.
abstract: The Fanconi anaemia (FA) FANCG protein is an integral component of the FA nuclear core complex that is required for monoubiquitylation of FANCD2. FANCG is also part of another protein complex termed D1-D2-G-X3 that contains FANCD2 and the homologous recombination repair proteins BRCA2 (FANCD1) and XRCC3. Formation of the D1-D2-G-X3 complex is mediated by serine-7 phosphorylation of FANCG and occurs independently of the FA core complex and FANCD2 monoubiquitylation. FANCG contains seven tetratricopeptide repeat (TPR) motifs that mediate protein-protein interactions and here we show that mutation of several of the TPR motifs at a conserved consensus residue ablates the in vivo binding activity of FANCG. Expression of mutated TPR1, TPR2, TPR5 and TPR6 in Chinese hamster fancg mutant NM3 fails to functionally complement its hypersensitivities to mitomycin C (MMC) and phleomycin and fails to restore FANCD2 monoubiquitylation. Using co-immunoprecipitation analysis, we demonstrate that these TPR-mutated FANCG proteins fail to interact with BRCA2, XRCC3, FANCA or FANCF. The interactions of other proteins in the D1-D2-G-X3 complex are also absent, including the interaction of BRCA2 with both the monoubiquitylated (FANCD2-L) and non-ubiquitylated (FANCD2-S) isoforms of FANCD2. Interestingly, a mutation of TPR7 (R563E), that complements the MMC and phleomycin hypersensitivity of human FA-G EUFA316 cells, fails to complement NM3, despite the mutated FANCG protein co-precipitating with FANCA, BRCA2 and XRCC3. Whilst interaction of TPR7-mutated FANCG with FANCF does appear to be reduced in NM3, FANCD2 is monoubiquitylated suggesting that sub-optimal interactions of FANCG in the core complex and the D1-D2-G-X3 complex are responsible for the observed MMC- and phleomycin-hypersensitivity, rather than a defect in FANCD2 monoubiquitylation. Our data demonstrate that FANCG functions as a mediator of protein-protein interactions and is vital for the assembly of multi-protein complexes including the FA core complex and the D1-D2-G-X3 complex.
authors.affiliation: Molecular Oncology and Stem Cell Research Group, School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: James B
authors.initials: JB
authors.lastname: Wilson
authors.forename: Eric
authors.initials: E
authors.lastname: Blom
authors.forename: Ryan
authors.initials: R
authors.lastname: Cunningham
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Xiao
authors.forename: Gary M
authors.initials: GM
authors.lastname: Kupfer
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Jones
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D024682:BRCA2 Protein / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D052241:Fanconi Anemia Complementation Group G Protein / Q000737:chemistry*; D006801:Humans; D051379:Mice; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D010692:Phleomycins / Q000494:pharmacology*; D011995:Recombination, Genetic; D014162:Transfection; D054875:Ubiquitination
chemical_list: D024682:BRCA2 Protein; D004268:DNA-Binding Proteins; C499905:FANCD2 protein, human; C499909:FANCG protein, human; D052236:Fanconi Anemia Complementation Group D2 Protein; D052241:Fanconi Anemia Complementation Group G Protein; D019384:Nucleic Acid Synthesis Inhibitors; D010692:Phleomycins; C110901:X-ray repair cross complementing protein 3
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