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Nigel J Jones - One of the best experts on this subject based on the ideXlab platform.
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several tetratricopeptide repeat tpr motifs of FANCG are required for assembly of the brca2 d1 d2 g x3 complex fancd2 monoubiquitylation and phleomycin resistance
Mutation Research, 2010Co-Authors: J B Wilson, Gary M Kupfer, Eric Blom, Yuxuan Xiao, Ryan Cunningham, Nigel J JonesAbstract: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.
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Several tetratricopeptide repeat (TPR) motifs of FANCG are required for assembly of the BRCA2/D1-D2-G-X3 complex, FANCD2 monoubiquitylation and phleomycin resistance
Mutation Research, 2010Co-Authors: J B Wilson, Gary M Kupfer, Eric Blom, Yuxuan Xiao, Ryan Cunningham, Nigel J JonesAbstract: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.
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Fanconi anemia complementation group FANCD2 protein serine 331 phosphorylation is important for fanconi anemia pathway function and BRCA2 interaction.
Cancer Research, 2009Co-Authors: J B Wilson, Nigel J Jones, Xiaoyong Chen, Diane S. Krause, Yuxuan Xiao, Gary M KupferAbstract:Fanconi anemia is a cancer-prone inherited bone marrow failure and cancer susceptibility syndrome with at least 13 complementation groups (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN). Our laboratory has previously described several regulatory phosphorylation events for core complex member proteins FANCG and FANCA by phosphorylation. In this study, we report a novel phosphorylation site serine 331 (S331) of FANCD2, the pivotal downstream player of the Fanconi anemia pathway. Phosphorylation of S331 is important for its DNA damage–inducible monoubiquitylation, resistance to DNA cross-linkers, and in vivo interaction with FANCD1/BRCA2. A phosphomimetic mutation at S331 restores all of these phenotypes to wild-type. In vitro and in vivo experiments show that phosphorylation of S331 is mediated by CHK1, the S-phase checkpoint kinase implicated in the Fanconi anemia DNA repair pathway. [Cancer Res 2009;69(22):8775–83]
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tetratricopeptide motif mediated interaction of FANCG with recombination proteins xrcc3 and brca2
DNA Repair, 2006Co-Authors: S. Hussain, Gary M Kupfer, P. Sung, L. H. Thompson, Eric Blom, Susan M Gordon, Hans Joenje, J B Wilson, Christopher G. Mathew, Nigel J JonesAbstract:Abstract Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1 , is the breast cancer susceptibility gene BRCA2 . At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG–XRCC3 and FANCG–BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein–protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.
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phosphorylation of fanconi anemia fa complementation group g protein FANCG at serine 7 is important for function of the fa pathway
Journal of Biological Chemistry, 2004Co-Authors: Fengyu Qiao, Jun Mi, Nigel J Jones, J B Wilson, Natalie R Bucheimer, Gary M KupferAbstract:Abstract Fanconi anemia (FA) is an autosomal recessive disease of cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA cross-linking agents. The molecular mechanism for the disease is unknown as few of the FA proteins have functional motifs. Several post-translational modifications of the proteins have been described. We and others (Qiao, F., Moss, A., and Kupfer, G. M. (2001) J. Biol. Chem. 276, 23391–23396 and Futaki, M., Watanabe, S., Kajigaya, S., and Liu, J. M. (2001) Biochem. Biophys. Res. Commun. 281, 347–351) have reported that the FANCG protein (Fanconi complementation group G) is phosphorylated. We show that in an in vitro kinase reaction FANCG is radioactively labeled. Mass spectrometry analysis detected a peptide containing phosphorylation of serine 7. Using PCR-mediated site-directed mutagenesis we mutated serine 7 to alanine. Only wild-type FANCG cDNA fully corrected FA-G mutant cells. We also tested the effect of human wild-type FANCG in Chinese hamster ovary cells in which the FANCG homologue is mutant. Human FANCG complemented these cells, whereas human FANCG(S7A) did not. Unexpectedly, FANCG(S7A) bound to and stabilized the endogenous forms of the FANCA and FANCC proteins in the FA-G cells. FANCG(S7A) aberrantly localized to globules in chromatin and did not abrogate the internuclear bridges seen in the FA-G mutant cells. Phosphorylation of serine 7 in FANCG is functionally important in the FA pathway.
J B Wilson - One of the best experts on this subject based on the ideXlab platform.
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Several tetratricopeptide repeat (TPR) motifs of FANCG are required for assembly of the BRCA2/D1-D2-G-X3 complex, FANCD2 monoubiquitylation and phleomycin resistance
Mutation Research, 2010Co-Authors: J B Wilson, Gary M Kupfer, Eric Blom, Yuxuan Xiao, Ryan Cunningham, Nigel J JonesAbstract: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.
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several tetratricopeptide repeat tpr motifs of FANCG are required for assembly of the brca2 d1 d2 g x3 complex fancd2 monoubiquitylation and phleomycin resistance
Mutation Research, 2010Co-Authors: J B Wilson, Gary M Kupfer, Eric Blom, Yuxuan Xiao, Ryan Cunningham, Nigel J JonesAbstract: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.
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Fanconi anemia complementation group FANCD2 protein serine 331 phosphorylation is important for fanconi anemia pathway function and BRCA2 interaction.
Cancer Research, 2009Co-Authors: J B Wilson, Nigel J Jones, Xiaoyong Chen, Diane S. Krause, Yuxuan Xiao, Gary M KupferAbstract:Fanconi anemia is a cancer-prone inherited bone marrow failure and cancer susceptibility syndrome with at least 13 complementation groups (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN). Our laboratory has previously described several regulatory phosphorylation events for core complex member proteins FANCG and FANCA by phosphorylation. In this study, we report a novel phosphorylation site serine 331 (S331) of FANCD2, the pivotal downstream player of the Fanconi anemia pathway. Phosphorylation of S331 is important for its DNA damage–inducible monoubiquitylation, resistance to DNA cross-linkers, and in vivo interaction with FANCD1/BRCA2. A phosphomimetic mutation at S331 restores all of these phenotypes to wild-type. In vitro and in vivo experiments show that phosphorylation of S331 is mediated by CHK1, the S-phase checkpoint kinase implicated in the Fanconi anemia DNA repair pathway. [Cancer Res 2009;69(22):8775–83]
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FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3
Oncogene, 2008Co-Authors: J B Wilson, A S Marriott, C G Mathew, P. Sung, Maureen E. Hoatlin, L. H. Thompson, M Takata, S. Hussain, K Yamamoto, Gary M KupferAbstract:Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2 , which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2–FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2–FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.
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tetratricopeptide motif mediated interaction of FANCG with recombination proteins xrcc3 and brca2
DNA Repair, 2006Co-Authors: S. Hussain, Gary M Kupfer, P. Sung, L. H. Thompson, Eric Blom, Susan M Gordon, Hans Joenje, J B Wilson, Christopher G. Mathew, Nigel J JonesAbstract:Abstract Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1 , is the breast cancer susceptibility gene BRCA2 . At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG–XRCC3 and FANCG–BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein–protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.
Henri J Van De Vrugt - One of the best experts on this subject based on the ideXlab platform.
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evidence for complete epistasis of null mutations in murine fanconi anemia genes fanca and FANCG
DNA Repair, 2011Co-Authors: Henri J Van De Vrugt, Mireille Koomen, Sietske T Bakker, Mariska Ad Berns, Yne De Vries, Martin A Rooimans, Ngan Ching Cheng, Martin Van Der Valk, Anneke B OostraAbstract:Abstract Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and FANCG to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a −/− / g −/− mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a −/− / g −/− mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a −/− / g −/− males and females. During the first year of life a −/− / g −/− did not develop malignancies or bone marrow failure. At the cellular level a −/− / g −/− , Fanca −/− , and FANCG −/− cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a −/− / g −/− double knockout mice and cells appeared highly similar to the phenotype of Fanca or FANCG single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or FANCG are fully epistatic, making additional important functions outside of the FA core complex highly unlikely.
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continuous in vivo infusion of interferon gamma ifn γ enhances engraftment of syngeneic wild type cells in fanca and FANCG mice
Blood, 2006Co-Authors: Yue Si, Henri J Van De Vrugt, Samantha L M Ciccone, Daisy Zeng, John Critser, Fengchun Yang, Fre Arwert, Shi Chen, Jin Yuan, Laura S HanelineAbstract:Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and FANCG–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.
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Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca- /- and FANCG- /- mice
Blood, 2006Co-Authors: Yue Si, Henri J Van De Vrugt, Samantha L M Ciccone, Daisy Zeng, John Critser, Fengchun Yang, Fre Arwert, Shi Chen, Jin Yuan, Laura S HanelineAbstract:Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and FANCG–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.
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hyperoxia induces defective epo induced signal transduction and loss of expansion capacity in fanca and FANCG deficient erythroid progenitors
Blood, 2004Co-Authors: Marieke Von Lindern, Henri J Van De Vrugt, Bob Löwenberg, Emile Van Den Akker, Fre Arwert, Godfrey Grech, Ivo P TouwAbstract:Fanconi anemia (FA) is characterized by chromosomal instability and bone marrow hypoplasia. To explore the mechanism of FA-related bone marrow failure, we studied erythropoietin (Epo)-dependent proliferation and differentiation of erythroid progenitors expanded from mice lacking the Fanca or FANCG gene. Erythroid progenitors can be expanded in serum-free medium supplemented with Epo, Stem Cell Factor (SCF) and glucocorticoids. The expansion capacity of Fanca −/− and FANCG −/− erythroid progenitors was reduced and the progenitors differentiated to mature erythrocytes instead after 10 days of culture. When Epo-induced signal transduction was compared between progenitors expanded for 6 days from fetal livers of Fanca −/− , FANCG −/− and wt littermates, Epo-induced phosphorylation of STAT5 and ERK1/2 appeared to be severely reduced and delayed in progenitors lacking Fanca or FANCG. Both in wt, Fanca −/− and FANCG −/− progenitors the phosphorylation of STAT5 and ERK1/2 increased with rising Epo-concentrations, but at low concentrations 4-fold more Epo was required to obtained comparable Stat5 phosphorylation in FancA −/− or FANCG −/− cells, while efficient phosphorylation at high concentrations was never achieved in Fanca or FANCG-deficient cells. As a consequence, the regulation of Epo-target genes like p21 WAF , Gilz, SOCS3, Spi2.1 and BclX L was impaired. In contrast to downstream signaling intermediates, Epo-induced phosphorylation of the Epo-receptor and its associated kinase JAK2 was not affected. Stem Cell Factor-induced c-Kit activation and downstream Erk1/2 phosphorylation was also equally efficient in wt, Fanca −/− and FANCG −/− progenitors. Thus, the signaling defect specifically seems to affect targets downstream of Jak2. Surprisingly, Epo-induced activation of Stat5 was unaffected in freshly isolated bone marrow cells. Comparison of Epo-induced Stat5 phosphorylation in erythroid progenitors cultured at atmospheric (20%) and physiologic (3%) oxygen revealed that Stat5 phosphorylation was only lost upon expansion of the progenitors at atmospheric (hyperoxic) conditions. Hyperoxia can damage many biomolecules among which DNA. In contrast to wt cells, Fanca −/− and FANCG −/− progenitors may not be able to repair DNA-damage induced by atmospheric oxygen. To examine whether increased DNA damage could impair Epo-induced signal transduction, we expanded erythroid progenitors from fetal livers of mice lacking the DNA-repair enzyme Ercc1. Ercc1 is directly involved in repair of DNA crosslinks, while a defective FA pathway renders cells hypersensitive to DNA-crosslinks. Epo-induced phosphorylation of Stat5 was impaired in Ercc1−/− progenitors, indicating that deficient repair of DNA damage, rather than FA-pathway specific mechanisms may cause the observed signaling defect. To substantiate this finding we treated wt and p53 −/− progenitors with increasing concentrations of mitomycin C (MMC). Sublethal concentrations of MMC abrogated the renewal capacity and impaired Epo-induced phosporylation of Stat5 and Erk1/2 in wt but not in p53 −/− erythroid progenitors. Together the data suggest that impaired progenitor expansion and Stat5 phosphorylation is not a specific aspect of cells lacking the fanconi pathway, but a general aspect of a p53-dependent response to genomic stress.
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multiple tpr motifs characterize the fanconi anemia FANCG protein
DNA Repair, 2004Co-Authors: Eric Blom, Henri J Van De Vrugt, Yne De Vries, Johan P. De Winter, Fre Arwert, Hans JoenjeAbstract:The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.
Fre Arwert - One of the best experts on this subject based on the ideXlab platform.
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Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors
The EMBO Journal, 2008Co-Authors: Karine Sii-felice, Fre Arwert, Olivier Etienne, Françoise Hoffschir, Céline Mathieu, Lydia Riou, Vilma Barroca, Céline Haton, Pierre Fouchet, François D. BoussinAbstract:Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and FANCG, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca−/− and FANCG−/− mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca−/− and FANCG−/− mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.
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continuous in vivo infusion of interferon gamma ifn γ enhances engraftment of syngeneic wild type cells in fanca and FANCG mice
Blood, 2006Co-Authors: Yue Si, Henri J Van De Vrugt, Samantha L M Ciccone, Daisy Zeng, John Critser, Fengchun Yang, Fre Arwert, Shi Chen, Jin Yuan, Laura S HanelineAbstract:Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and FANCG–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.
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Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca- /- and FANCG- /- mice
Blood, 2006Co-Authors: Yue Si, Henri J Van De Vrugt, Samantha L M Ciccone, Daisy Zeng, John Critser, Fengchun Yang, Fre Arwert, Shi Chen, Jin Yuan, Laura S HanelineAbstract:Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and FANCG–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.
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hyperoxia induces defective epo induced signal transduction and loss of expansion capacity in fanca and FANCG deficient erythroid progenitors
Blood, 2004Co-Authors: Marieke Von Lindern, Henri J Van De Vrugt, Bob Löwenberg, Emile Van Den Akker, Fre Arwert, Godfrey Grech, Ivo P TouwAbstract:Fanconi anemia (FA) is characterized by chromosomal instability and bone marrow hypoplasia. To explore the mechanism of FA-related bone marrow failure, we studied erythropoietin (Epo)-dependent proliferation and differentiation of erythroid progenitors expanded from mice lacking the Fanca or FANCG gene. Erythroid progenitors can be expanded in serum-free medium supplemented with Epo, Stem Cell Factor (SCF) and glucocorticoids. The expansion capacity of Fanca −/− and FANCG −/− erythroid progenitors was reduced and the progenitors differentiated to mature erythrocytes instead after 10 days of culture. When Epo-induced signal transduction was compared between progenitors expanded for 6 days from fetal livers of Fanca −/− , FANCG −/− and wt littermates, Epo-induced phosphorylation of STAT5 and ERK1/2 appeared to be severely reduced and delayed in progenitors lacking Fanca or FANCG. Both in wt, Fanca −/− and FANCG −/− progenitors the phosphorylation of STAT5 and ERK1/2 increased with rising Epo-concentrations, but at low concentrations 4-fold more Epo was required to obtained comparable Stat5 phosphorylation in FancA −/− or FANCG −/− cells, while efficient phosphorylation at high concentrations was never achieved in Fanca or FANCG-deficient cells. As a consequence, the regulation of Epo-target genes like p21 WAF , Gilz, SOCS3, Spi2.1 and BclX L was impaired. In contrast to downstream signaling intermediates, Epo-induced phosphorylation of the Epo-receptor and its associated kinase JAK2 was not affected. Stem Cell Factor-induced c-Kit activation and downstream Erk1/2 phosphorylation was also equally efficient in wt, Fanca −/− and FANCG −/− progenitors. Thus, the signaling defect specifically seems to affect targets downstream of Jak2. Surprisingly, Epo-induced activation of Stat5 was unaffected in freshly isolated bone marrow cells. Comparison of Epo-induced Stat5 phosphorylation in erythroid progenitors cultured at atmospheric (20%) and physiologic (3%) oxygen revealed that Stat5 phosphorylation was only lost upon expansion of the progenitors at atmospheric (hyperoxic) conditions. Hyperoxia can damage many biomolecules among which DNA. In contrast to wt cells, Fanca −/− and FANCG −/− progenitors may not be able to repair DNA-damage induced by atmospheric oxygen. To examine whether increased DNA damage could impair Epo-induced signal transduction, we expanded erythroid progenitors from fetal livers of mice lacking the DNA-repair enzyme Ercc1. Ercc1 is directly involved in repair of DNA crosslinks, while a defective FA pathway renders cells hypersensitive to DNA-crosslinks. Epo-induced phosphorylation of Stat5 was impaired in Ercc1−/− progenitors, indicating that deficient repair of DNA damage, rather than FA-pathway specific mechanisms may cause the observed signaling defect. To substantiate this finding we treated wt and p53 −/− progenitors with increasing concentrations of mitomycin C (MMC). Sublethal concentrations of MMC abrogated the renewal capacity and impaired Epo-induced phosporylation of Stat5 and Erk1/2 in wt but not in p53 −/− erythroid progenitors. Together the data suggest that impaired progenitor expansion and Stat5 phosphorylation is not a specific aspect of cells lacking the fanconi pathway, but a general aspect of a p53-dependent response to genomic stress.
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The Fanconi anemia gene product FANCF is a flexible adaptor protein.
Journal of Biological Chemistry, 2004Co-Authors: Eric Blom, Quinten Waisfisz, Martin A Rooimans, Alexandra Sobeck, Fre Arwert, Annette L. Medhurst, Patrick Bier, El Houari Laghmani, Mark H. Johnson, K. J. PatelAbstract:The Fanconi anemia (FA) protein FANCF is an essential component of a nuclear core complex that protects the genome against chromosomal instability, but the specific function of FANCF is still poorly understood. Based upon the homology between human and Xenopus laevis FANCF, we carried out an extensive mutagenesis study to examine which domains are functionally important and to gain more insight into the function of FANCF. In contrast to previous suggestions, we show that FANCF does not have a ROM-like function. We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex. The N terminus appears to stabilize the interaction with FANCA and FANCG and is essential for the binding of the FANCC/FANCE subcomplex. We identified several important amino acids in this N-terminal region but, surprisingly, many amino acid changes failed to affect the function of the FANCF protein. Our data demonstrate that FANCF acts as a flexible adaptor protein that plays a key role in the proper assembly of the FA core complex.
Alan D Dandrea - One of the best experts on this subject based on the ideXlab platform.
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heterogeneous activation of the fanconi anemia pathway by patient derived fanca mutants
Human Molecular Genetics, 2002Co-Authors: Daiki Adachi, Toshiyasu Taniguchi, Keiko Nakasato, Shigetaka Asano, Hiroshi Yagasaki, Alan D Dandrea, Takayuki YamashitaAbstract:Fanconi anemia (FA) is an autosomal recessive disorder of hematopoiesis characterized by hypersensitivity to DNA crosslinkers such as mitomycin C (MMC). There is growing evidence for a model of the FA pathway, wherein a nuclear multiprotein complex of five FA proteins (FANCA, C, E, F and G) regulates activation of FANCD2 into a monoubiquitinated form, which, collaborating with the BRCA1 machinery, affects cellular response to DNA damage. However, the role of the FA pathway in defective DNA damage response caused by various mutant forms of FA proteins has not been fully assessed. In the present study, 21 patient-derived FANCA mutants with a missense or a small in-frame deletion were expressed in FANCA-deficient fibroblasts and examined for complementation of MMC sensitivity and for reconstitution of the FA pathway: FANCA phosphorylation, interaction with FANCC, FANCF and FANCG and nuclear localization and FANCD2 monoubiquitination. The altered FANCA proteins complemented MMC sensitivity at different grades: five proteins (group I) behaved like wild-type FANCA, whereas the other proteins were either mildly (group II, n=4) or severely (group III, n = 12) impaired. Group I proteins showed an apparently normal reconstitution of the FA pathway, thus they may be pathogenic by reducing endogenous expression or possibly benign polymorphisms. Reconstitution of the FA pathway by group II and III mutants closely correlated with cellular sensitivity to MMC. The different activation of the FA pathway may partly account for the phenotypic variation seen in FA patients.
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brca1 interacts directly with the fanconi anemia protein fanca
Human Molecular Genetics, 2002Co-Authors: Alexandra Folias, Mara Matkovic, Donald A Bruun, Sonja Reid, James Hejna, Markus Grompe, Alan D Dandrea, Robb E MosesAbstract:Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740–1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.
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a cytoplasmic serine protein kinase binds and may regulate the fanconi anemia protein fanca
Blood, 2001Co-Authors: Hiroshi Yagasaki, Irene Garciahiguera, Daiki Adachi, Makoto Futaki, Nii Tetteh, Shigetaka Asano, Alan D Dandrea, Takayuki YamashitaAbstract:Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FAgenes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA− cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.
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the chinese hamster FANCG xrcc9 mutant nm3 fails to express the monoubiquitinated form of the fancd2 protein is hypersensitive to a range of dna damaging agents and exhibits a normal level of spontaneous sister chromatid exchange
Carcinogenesis, 2001Co-Authors: J B Wilson, Alan D Dandrea, Mark A Johnson, Anna Stuckert, Kelly L Trueman, Peter E Bryant, Raymond E Meyn, Nigel J JonesAbstract:Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustardhypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein’s subsequent interaction with
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targeted disruption of the murine fanconi anemia gene FANCG xrcc9
Blood, 2001Co-Authors: Yi Yang, Yanan Kuang, Tobias Hays, Lisa A Moreau, Naifang Lu, Brian Seed, Alan D DandreaAbstract:Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common cellular pathway. To further understand the in vivo role of one of these human genes (FANCG), we generated a targeted disruption of murine FANCG and bred mice homozygous for the targeted allele. Similar to the phenotype of the previously described Fancc −/− and Fanca −/− mice, the FANCG −/− mice had normal viability and no gross developmental abnormalities. Primary splenic lymphocytes, bone marrow progenitor cells, and murine embryo fibroblasts from the FANCG −/− mice demonstrated spontaneous chromosome breakage and increased sensitivity to mitomycin C and, to a lesser extent, ionizing radiation. FANCG −/− lymphocytes had a defect in the FA pathway, based on their failure to activate the monoubiquitination of the downstream Fancd2 protein in response to IR. Finally, FANCG −/− mice had decreased fertility and abnormal gonadal histology. In conclusion, disruption of the FANCG gene confirms the role of FANCG in the FA pathway. The FANCG −/− mouse may be useful as an animal model for future gene therapy and cancer susceptibility studies.
