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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, Ryan Cunningham, Yuxuan Xiao, 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: James B Wilson, Gary M Kupfer, Eric Blom, Ryan Cunningham, Yuxuan Xiao, 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, Yuxuan Xiao, Diane S. Krause, Xiaoyong Chen, 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, C G Mathew, L. H. Thompson, Eric Blom, Susan M Gordon, Hans Joenje, P. Sung, J. B. Wilson, 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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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, Kelly L Trueman, Raymond E Meyn, A. Stuckert, Mark A Johnson, Peter E. Bryant, 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
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, Ryan Cunningham, Yuxuan Xiao, 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, Yuxuan Xiao, Diane S. Krause, Xiaoyong Chen, 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, Maureen E. Hoatlin, L. H. Thompson, M. Takata, S. Hussain, P. Sung, 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, C G Mathew, L. H. Thompson, Eric Blom, Susan M Gordon, Hans Joenje, P. Sung, J. B. Wilson, 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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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, Kelly L Trueman, Raymond E Meyn, A. Stuckert, Mark A Johnson, Peter E. Bryant, 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
Gary M Kupfer - One of the best experts on this subject based on the ideXlab platform.
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Regulation of PCNA Mono-Ubiqutination by FANCD2 and Rad51 in Response to Hydroxyurea.
Blood, 2012Co-Authors: Gary M Kupfer, Xiaoyong ChenAbstract:Abstract 2377 FANCD2 is a key player in FA pathway. It has been shown that FANCD2 can interact with PCNA and with Rad18, the ubiquitin ligase responsible for PCNA mono-ubiquitination. The mono-ubiquitination of PCNA is very important for its function in translesion synthesis. We found that in response to DNA damage agent hyxdroxyurea (HU) the interaction of FANCD2 with Rad18 or Rad51, and the interaction of Rad51 with Rad18 or PCNA was enhanced. FANCD2 is required for increased interaction between Rad51 and Rad18 indicating that FANCD2, Rad51 and Rad18 form a complex in response to HU. Rad18 was required for PCNA mono-ubiquitination in response to HU. FANCD2 deficient cells failed to enhance the interaction between Rad18 and Rad51. Furthermore, PCNA mono-ubiquitination was impaired in FANCD2 deficient cells in response to HU. FANCD2 mono-ubiquitination deficient mutant partially rescued PCNA mono-ubiquitination. The partial mono-ubiquitination of PCNA in response to HU in FANCA deficient mutant confirmed the role of non-ubiquitinated FANCD2 in PCNA mono-ubiquitination. The normal mono-ubiquitination of PCNA in FANCJ deficient mutant confirmed that the effect of FANCD2 in PCNA mon-ubiquitination is not due to FA pathway deficiency. Rad51 was also involved in regulating PCNA mono-ubiquitination in response to HU. Rad51 siRNA knock down cells showed decreased PCNA mono-ubiquitination in response to HU. The role of Rad51 in regulating PCNA mono-ubiquitination did not require BRCA1, indicating that this function is independent of HR. More importantly FANCD2 deficient cells were hypersensitive to HU, whereas FANCD2 mono-ubiquitination deficient mutant cells, FANCD2 corrected cells, FANCA deficient cells and FANCJ deficient cells were not hypersensitive to HU. Our data indicate that FANCD2 plays an important role in PCNA mono-ubiquitination and translesion synthesis partially in a mono-ubiquitination independent manner. Rad51 also plays an important role in PCNA mono-ubiquitination and translesion synthesis in a HR independent fashion. Disclosures: No relevant conflicts of interest to declare.
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The E3 ubiquitin ligase RAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI
Blood, 2011Co-Authors: Stacy A. Williams, Simonne Longerich, Patrick Sung, Cyrus Vaziri, Gary M KupferAbstract:Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.
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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, Ryan Cunningham, Yuxuan Xiao, 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: James B Wilson, Gary M Kupfer, Eric Blom, Ryan Cunningham, Yuxuan Xiao, 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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Purification of FANCD2 sub-complexes.
British journal of haematology, 2010Co-Authors: Gang Zhi, Xiaoyong Chen, William W. Newcomb, Jay C. Brown, Oliver John Semmes, Gary M KupferAbstract:Fanconi anaemia (FA) is a recessive genetic disorder characterized by bone marrow failure, birth defects and cancer. Cells from FA patients are particularly defective in removing DNA interstrand crosslinks. We have developed a working chromatography purification scheme for FANCD2, a pivotal player in the FA DNA repair pathway, to facilitate identification of FANCD2 interacting partners. In doing so, at least three distinct FANCD2 subcomplexes were found to be present, designated as large, middle, and small complexes. The small complex is composed of tetramer of FANCD2 polypeptides, which may be the building block for other FANCD2 subcomplexes.
Alan D. D'andrea - One of the best experts on this subject based on the ideXlab platform.
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FANCD2-FANCI Hurdles the DNA Interstrand Crosslink
Cell, 2009Co-Authors: George-lucian Moldovan, Alan D. D'andreaAbstract:Like good housekeepers, cells spend a lot of energy caring for their genetic material, mopping up DNA damage that may alter the reading of their genetic information. The most difficult to repair, and at the same time the most detrimental, are the DNA interstrand crosslinks (ICLs). Since ICLs block DNA replication, their removal is an essential requirement for cell survival. The severe clinical symptoms of patients with Fanconi Anemia (blood marrow failure, developmental abnormalities, and cancer predisposition, often leading to an early death) testify to the toxicity of ICLs (Moldovan and D'Andrea, 2009; Patel and Joenje, 2007). How the FA pathway protects against ICLs has long remained a mystery. Using a cell-free system, Knipscheer et al (Knipscheer et al., 2009) now show, for the first time, that FA proteins are directly involved, at several steps, in the ICL repair process. Removal of ICLs occurs mostly in S-phase and involves the stepwise involvement of nucleases, specialized lesion bypass DNA polymerases, and homologous recombination factors (Moldovan and D'Andrea, 2009). Using the tractable cell-free replication system of Xenopus egg extracts, the authors previously devised an experimental approach to investigate replication-dependent ICL repair (Raschle et al., 2008). As illustrated in Figure 1, their system reveals that replication forks, converging from both directions, initially stop 20-40 nucleotides from the crosslink. One of the forks subsequently moves further, and stops again just before the crosslink (at position -1). Nucleolytic incisions on both sides of the crosslink, and DNA polymerization across the lesion, restore one of the chromatids, while the other is most likely repaired through homologous recombination. In an elegant application of their crosslink repair system, Knipscheer et al now show that loss of the FA pathway can block both the incision and bypass steps. Figure 1 FANCD2 monoubiquitination is required for the nucleolytic incision and translesion bypass steps of interstrand crosslink repair Thirteen FA proteins cooperate in the FA pathway. Eight of these proteins form an ubiquitin ligase complex that monoubiquitinates the substrates FANCD2 and FANCI. In the new study, immunodepletion of FANCD2 from the egg extracts (which also co-depletes its heterodimeric partner FANCI), dramatically inhibited crosslink repair, demonstrating that FA is a true DNA repair syndrome. Nucleotide insertion opposite the ICL was blocked in the absence of FANCD2, since the leading strand progressed only to position -1. Also, the two incisions required to unhook the crosslink could not be detected in extracts depleted of FANCD2. All defects could be rescued by adding back the recombinant FANCD2-FANCI complex, but not by adding back a complex containing a point mutant of FANCD2 that cannot by ubiquitinated. Accordingly, when the investigators examined the timing of FANCD2 ubiquitination, they found that this modification occurs precisely when the replication fork reaches the -1 position. FANCD2 ubiquitination, known to be essential for crosslink tolerance, is therefore required to advance the replication fork across the crosslink, by orchestrating crosslink unhooking and translesion synthesis. Bypass of DNA lesions is a potentially mutagenic process performed by specialized polymerases. The FA pathway is known to be required for mutagenesis, and the polymerase Rev1 is epistatic to FA, and may function in FA-dependent lesion bypass (Mirchandani et al., 2008; Niedzwiedz et al., 2004). The finding that FANCD2 ubiquitination promotes crosslink bypass supports a model in which Rev1 is recruited, via its ubiquitin binding domain, to the FANCD2-Ub protein. This model is analogous to the recruitment of Y-family polymerases, such as DNA polymerase η, by monoubiquitinated PCNA in order to bypass UV-induced lesions (Bienko et al., 2005). Rev1 depletion from egg extracts, together with Rev1 interaction studies, should now be able to directly test this hypothesis. Since human cells have at least fifteen polymerases, a different mutagenic polymerase might also be involved. Interestingly, only about 25% of crosslinked plasmids are recovered as repaired, error-free DNA molecules (Knipscheer et al., 2009), raising the possibility that some sites are repaired with errors. Investigating the mutation spectrum of the repaired templates may help identify the relevant polymerases. Alternatively, FANCD2 may not directly recruit a polymerase, but rather stabilize some structure at the crosslink site, thus indirectly allowing a nucleotide insertion event. How the FANCD2 protein regulates the DNA incisions flanking the crosslink is even more intriguing. FANCD2 has no known enzymatic activity, possesses no obvious nuclease domains, and has no known associated nucleases. Perhaps a cryptic nuclease activity of FANCD2 is activated by monoubiquitination when a replication fork encounters a crosslink. Alternatively, the FANCD2-Ub may transiently recruit some known nuclease complex, such as the Mus81-Eme1, Xpf-Ercc1, or the newly-described Slx4 complex. At present, the Xenopus experimental system cannot temporally dissociate the incision and bypass steps. Although it is logical to assume that incision occurs first, allowing bypass by a polymerase such as Rev1, the alternative possibility should not be excluded: nucleotide insertion opposite the ICL may create a DNA structure that promotes incision. Also, it is possible that the proximal incision occurs first, followed by the lesion bypass step and the distal incision. In either case, FANCD2 may only be required for the first activity in the stepwise process. Depletion of FANCD2 may thus only indirectly affect the subsequent steps. Alternatively, FANCD2 may directly activate both incision and bypass, irrespective of their order of occurrence. Recent studies indicate that the regulated deubiquitination of FANCD2 by the ubiquitin protease complex, USP1/UAF1, is also required for crosslinker tolerance (Moldovan and D'Andrea, 2009). Whether deubiquitination is required for incision and/or lesion bypass is also unknown. The Xenopus system may be useful in resolving these questions as well. An apparent shortcoming of this model system is its inability (to date) to decipher the roles of the FA pathway in the last part of the ICL repair process -namely, the homologous recombination (HR) step. Only the repair of the chromatid with the unhooked crosslink can be investigated, probably due to degradation of the other sister chromatid that is broken by incisions. Therefore, the repair process in this system terminates before the final recombination step. It is possible that FANCD2 plays only an indirect role in the HR step, simply “handing off” the product of the incision and insertion steps to the HR machinery. This most likely involves the recombination mediator BRCA2, identified as the FA factor FANCD1 (Howlett et al., 2002), and its binding partner, FANCN. Alternatively, the FA pathway may be actively involved in HR. This possibility is supported by evidence that FANCD2 and FANCI are required for double strand break-induced HR repair (Smogorzewska et al., 2007), suggesting a general role of the FA pathway in promoting recombination. Whether this function represents a direct participation (for example, recruitment of HR factors), or a more indirect one, such as inhibition of alternative competing repair pathways (such as the error prone non-homologous end joining pathway), is currently unclear. Despite these shortcomings, Xenopus egg extracts should allow the identification and characterization of the nucleases and polymerases involved in ICL repair, and elucidate how these activities are controlled by the FA pathway. This knowledge may, over time, lead to better therapeutic strategies for FA patients. Together with the recently described role of the FA pathway in crosslink-induced checkpoint signaling (Ben-Yehoyada et al., 2009), these new findings paint the picture of a dynamic, multileveled cellular pathway that is involved at multiple steps in the DNA crosslink response.
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Functional Interaction between FANCD2 and ATM in the DNA Damage Response.
Blood, 2005Co-Authors: Steven P. Margossian, Richard D. Kennedy, Alan D. D'andreaAbstract:Fanconi Anemia is a multigenic cancer susceptibility syndrome. The FA pathway controls the monoubiquitination of FANCD2, and its targeting to damage-associated DNA foci. Cells that are deficient in the FA pathway are highly sensitive to bi-functional crosslinking agents such as mitomycin-C (MMC). Although FANCD2 monoubiquitination is activated by DNA damage, how this is coupled to the DNA damage response is unknown. Previous studies suggest a functional interaction between FANCD2 and the protein kinase ATM (ataxia telangiectasia mutated). Recently we have shown that the C-terminus of FANCD2 is required for its function (Montes de Oca et al., Blood2005:105, 1003). A C-terminal truncation of FANCD2 (FANCD2-C) fails to correct the MMC sensitivity FANCD2 −/− cells despite its monoubiquitination and assembly into DNA damage foci. In the present study we demonstrate that FANCD2 forms a protein complex with the ATM kinase and that this interaction requires the FANCD2 C-terminus. The association of ATM with FANCD2 occurs in the absence of DNA damage, and does not require FANCD2 to be monoubiquitinated. In immunoprecipitation experiments involving the FANCD2-C mutant, ATM fails to co-precipitate, suggesting the C-terminus of FANCD2 is required for recruiting ATM to sites of DNA damage. In addition, we have identified a new ATM phosphorylation site on FANCD2, Thr 691 . Using a phospho-specific antibody to FANCD2 Thr 691 we have determined that siRNA knockdown of ATM results in loss of phosphorylation of Thr 691 , and ionizing radiation (IR) activates the phosphorylation of Thr 691 . Mutation of Thr 691 to Alanine destroys this ATM phosphorylation site. Interestingly, this FANCD2 mutant also fails to correct the MMC sensitivity FANCD2 −/− cells even though it can be monoubiquitinated and targeted to DNA foci following DNA damage, a phenotype observed for the FANCD2-C mutant. Next we examined the phosphorylation of Thr 691 in FANCD2-C. In FANCD2 constructs expressing a mutated or truncated C-terminus, phosphorylation of Thr 691 is delayed following exposure to MMC and the cells are sensitive to DNA damage. Following exposure to ionizing radiation the ATM kinase phosphorylates itself on Ser 1981 . Using a phospho-specific antibody to ATM Ser 1981 we evaluated the phosphorylation of ATM in response to DNA damage caused by IR and MMC. Western analysis shows that phosphorylation of ATM on Ser 1981 following IR is the same in FANCD2 and FANCD2-C cells as well as uncorrected FAND2 −/− cells. ATM foci formation following IR also appears identical in all three FANCD2 cell lines based upon immunoflourescence microscopy. However in cells damaged with MMC, there is a delay in Ser 1981 phosphorylation and fewer ATM foci in the uncorrected and FANCD2-C cells compared to cells corrected with intact FANCD2. We propose a model for FANCD2 function where the targeting of FANCD2 to DNA damage inducible foci is required for the proper coordination of some ATM-dependent DNA repair and checkpoint responses. For instance, FANCD2 may be required for targeting ATM to a subset of double strand breaks - namely, those breaks associated with an adjacent interstrand crosslink. Thus targeting of ATM to MMC-induced DNA damage requires FANCD2 but recruitment to IR-induced double strand breaks does not, depending instead on NBS1 and the M/R/N complex.
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Regulation of the Fanconi Anemia/BRCA pathway
Cancer Research, 2005Co-Authors: Alan D. D'andreaAbstract:Proc Amer Assoc Cancer Res, Volume 46, 2005 SY28-2 Fanconi Anemia (FA) is an autosomal recessive cancer susceptibility syndrome characterized by cellular hypersensitivity to DNA crosslinking agents. There are eleven complementation groups of FA (A, B, C, D1, D2, E, F, G, L, I, J), and nine FA genes have been cloned. The FANCD2 gene is identical to the BRCA2 gene. The nine cloned FA proteins cooperate in a novel cellular DNA damage response pathway (the FA/BRCA pathway). This pathway is activated during the S phase of the cell cycle. Specifically, in S phase, when a replication fork encounters an interstrand crosslink, the FA complex (consisting of FA proteins A, B, C, E, F, G, L) becomes an active monoubiquitin ligase. This complex monoubiquitinates the downstream FANCD2 protein, leading to its targeting to DNA repair foci containing FANCD1/BRCA2. Disruption of this pathway leads to a failure to repair DNA crosslinks. We have recently identified other regulatory proteins which control the cell cycle specific activation of the FA/BRCA pathway. The ATR kinase, for example, is required for the S phase specific phosphorylation of FANCD2 (Andreassen PR, D’Andrea AD, Taniguchi T. ATR couples FANCD2 monoubiquitination to the DNA-damage response. Genes & Development 18: 1958-1963, 2004). In addition, we have identified a novel deubiquitinating enzyme (USP1) which cleaves FANCD2-Ub (Nijman et al, 2004, submitted). FANCD2-Ub is localized to the chromatin, has DNA binding activity, and appears to be required to prevent collapse of the replication fork. USP1 cleavage of FANCD2-Ub at the end of S phase, results in exit of FANCD2 from the chromatin fraction and a completion of DNA replication. While the precise role of FANCD2 monoubiquitination during S phase is unknown, it may function as a mechanism to (a) turn on homologous recombination (HR) repair and/or (b) switch on DNA translesion synthesis (TLS) polymerases. The precise role of the USP1 protease in regulation the of the FA/BRCA pathway will be discussed.
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Regulated interaction of the Fanconi anemia protein, FANCD2, with chromatin.
Blood, 2004Co-Authors: Rocío Montes De Oca, Toshiyasu Taniguchi, Markus Grompe, Scott Houghtaling, Xiaozhe Wang, Paul R. Andreassen, Steven P. Margossian, Richard C. Gregory, Alan D. D'andreaAbstract:DNA damage activates the monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, resulting in the assembly of FANCD2 nuclear foci. In the current study, we characterize structural features of FANCD2 required for this intranuclear translocation. We have previously identified 2 normal mRNA splice variants of FANCD2, one containing exon 44 sequence at the 3′ end (FANCD2-44) and one containing exon 43 sequence (FANCD2-43). The 2 predicted FANCD2 proteins differ in their carboxy terminal 24 amino acids. In stably transfected FANCD2—/— fibroblasts, FANCD2-44 and FANCD2-43 proteins were monoubiquitinated on K561. Only FANCD2-44 corrected the mitomycin C (MMC) sensitivity of the transfected cells. We find that monoubiquitinated FANCD2-44 was translocated from the soluble nuclear compartment into chromatin. A mutant form of FANCD2-44 (FANCD2-K561R) was not monoubiquitinated and failed to bind chromatin. A truncated FANCD2 protein (Exon44-T), lacking the carboxy terminal 24 amino acids encoded by exon 44 but retaining K561, and another mutant FANCD2 protein, with a single amino acid substitution at a conserved residue within the C-terminal 24 amino acids (D1428A), were monoubiquitinated. Both mutants were targeted to chromatin but failed to correct MMC sensitivity. Taken together, our results indicate that monoubiquitination of FANCD2 regulates chromatin binding and that D1428 within the carboxy terminal acidic sequence encoded by exon 44 is independently required for functional complementation of FA-D2 cells. We hypothesize that the carboxy terminus of FANCD2-44 plays a critical role in sensing or repairing DNA damage.
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Functional Interaction of Monoubiquitinated FANCD2 and BRCA2/FANCD1 in Chromatin
Molecular and cellular biology, 2004Co-Authors: Xiaozhe Wang, Paul R. Andreassen, Alan D. D'andreaAbstract:Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2−/− cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2−/− cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.
Alexandra Sobeck - One of the best experts on this subject based on the ideXlab platform.
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FANCD2 fancj and brca2 cooperate to promote replication fork recovery independently of the fanconi anemia core complex
Cell Cycle, 2015Co-Authors: Maya Raghunandan, Indrajit Chaudhury, Stephanie L Kelich, Helmut Hanenberg, Alexandra SobeckAbstract:Fanconi Anemia (FA) is an inherited multi-gene cancer predisposition syndrome that is characterized on the cellular level by a hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA pathway proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex monoubiquitinates the central FA pathway members FANCD2 and FANCI, followed by their recruitment to chromatin. Chromatin-bound monoubiquitinated FANCD2 and FANCI subsequently coordinate DNA repair factors including the downstream FA pathway members FANCJ and FANCD1/BRCA2 to repair the DNA ICL. Importantly, we recently showed that FANCD2 has additional independent roles: it binds chromatin and acts in concert with the BLM helicase complex to promote the restart of aphidicolin (APH)-stalled replication forks, while suppressing the firing of new replication origins. Here, we show that FANCD2 fulfills these roles independently of the FA core complex-mediated monoubiquitination step. Following APH treatment, nonubiquitinated FANCD2 accumulates on chromatin, recruits the BLM complex, and promotes robust replication fork recovery regardless of the absence or presence of a functional FA core complex. In contrast, the downstream FA pathway members FANCJ and BRCA2 share FANCD2's role in replication fork restart and the suppression of new origin firing. Our results support a non-linear FA pathway model at stalled replication forks, where the nonubiquitinated FANCD2 isoform – in concert with FANCJ and BRCA2 – fulfills a specific function in promoting efficient replication fork recovery independently of the FA core complex.
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CtIP mediates replication fork recovery in a FANCD2-regulated manner
Human molecular genetics, 2014Co-Authors: Jung Eun Yeo, Eu Han Lee, Eric A. Hendrickson, Alexandra SobeckAbstract:Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Within the FA pathway, an upstream FA core complex mediates monoubiquitination and recruitment of the central FANCD2 protein to sites of stalled replication forks. Once recruited, FANCD2 fulfills a dual role towards replication fork recovery: (i) it cooperates with BRCA2 and RAD51 to protect forks from nucleolytic degradation and (ii) it recruits the BLM helicase to promote replication fork restart while suppressing new origin firing. Intriguingly, FANCD2 and its interaction partners are also involved in homologous recombination (HR) repair of DNA double-strand breaks, hinting that FANCD2 utilizes HR proteins to mediate replication fork recovery. One such candidate is CtIP (CtBP-interacting protein), a key HR repair factor that functions in complex with BRCA1 and MRE11, but has not been investigated as putative player in the replication stress response. Here, we identify CtIP as a novel interaction partner of FANCD2. CtIP binds and stabilizes FANCD2 in a DNA damage- and FA core complex-independent manner, suggesting that FANCD2 monoubiquitination is dispensable for its interaction with CtIP. Following cellular treatment with a replication inhibitor, aphidicolin, FANCD2 recruits CtIP to transiently stalled, as well as collapsed, replication forks on chromatin. At stalled forks, CtIP cooperates with FANCD2 to promote fork restart and the suppression of new origin firing. Both functions are dependent on BRCA1 that controls the step-wise recruitment of MRE11, FANCD2 and finally CtIP to stalled replication forks, followed by their concerted actions to promote fork recovery.
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Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase
Nucleic acids research, 2012Co-Authors: Archana Sareen, Indrajit Chaudhury, Nicole Adams, Alexandra SobeckAbstract:Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2-FANCI complex versus the monomeric proteins are. We show that the FANCD2-FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2-FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to-and independently of-FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase.
