Oxidative Stress/Damage Induces Multimerization and Interaction of Fanconi Anemia Proteins

Abstract

Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function. Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function. Fanconi anemia (FANC) 1The abbreviations used are: FANC, Fanconi anemia; MMC, mitomycin C; DTT, dithiothreitol; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase.1The abbreviations used are: FANC, Fanconi anemia; MMC, mitomycin C; DTT, dithiothreitol; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase. is an autosomal recessive disorder characterized by chromosomal instability and defective DNA repair, and FANC-deficient cells exhibit extreme sensitivity toward oxygen and DNA-cross-linking agents such as diepoxybutane and mitomycin C (1Joenje H. Oostra A.B. Wijker M. di Summa F.M. van Berkel C.G. Rooimans M.A. Ebell W. van Weel M. Pronk J.C. Buchwald M. Arwert F. Am. J. Hum. Genet. 1997; 61: 940-944Abstract Full Text PDF H. Genet. M. H. H. of of FANC have been identified and (FANCA, and (1Joenje H. Oostra A.B. Wijker M. di Summa F.M. van Berkel C.G. Rooimans M.A. Ebell W. van Weel M. Pronk J.C. Buchwald M. Arwert F. Am. J. Hum. Genet. 1997; 61: 940-944Abstract Full Text PDF H. Genet. M. H. H. in of the genes lead to FANC a of genetic with that of DNA that a and FANCG proteins are part of a nuclear complex their and the of complex results in the and to FANC J. FANCA a and with in the H. FANCA cells a FANC defective in their and to to a FANCA to the mitomycin C sensitivity of that FANCA may be in nuclear of function in repair. FANCG a that been identified as the that is to agents, and DNA-cross-linking agents M. van 1997; cells also show a of chromosomal that be by of 1997; of the of FANC proteins in DNA by the interaction of with following DNA damage J. M. Full Text Full Text PDF is to and is FANC genes in that is the of in response to F. H. that may be in FANC a following H2O2 a FANC proteins in and of oxidative DNA J. H. Buchwald M. M. J. H. J. Full Text Full Text PDF and not between FANCA and FANCG by and J. J. van Weel Arwert F. H. H. F. H. H. J. Full Text Full Text PDF H. H. C.G. Buchwald M. the of FANC proteins have to be is a the FANC proteins in DNA and damage we the interaction of FANC proteins following oxidative that FANCA and FANCG are multimerized and/or form a complex via intermolecular disulfide in response to oxidative as as interaction with of FANCD2 H2O2 that interaction may be an response to oxidative damage that a crucial in DNA and damage and and in with FANCA and and FANCG and and and and and H2O2 of FANCA and FANCG FANCG by the and the Both a and the also a and by DNA FANCA at and by the and by and DNA cells FANCG at by the and the by and DNA and of cells and cells in and which with and in cells in with H2O2 at the at cells with and the by of in in the of and to and with a at and the with and with and proteins by used in to the at at least with that been in with in the the with of proteins in cells following J. and by and by a M. J. Full Text Full Text PDF and proteins under under to a and with an with an by by the by in a at at and with at to a of and in and with at by at and the by with of and by as the in the of of and by and of FANC under to a FANC proteins in and the of oxidative H. with cells FANC genes hypersensitivity to H2O2 treatment H. J. Full Text Full Text PDF and not FANC proteins (FANCA, and of we of in of FANC cells with FANCG by following a and the of as multimers in under oxidizing conditions, whereas as a with of to the of intermolecular disulfide FANCG but not the of the not form multimers of is to that form of multimers in a to a under oxidizing and of of FANCA and FANCG in a of FANC cells with a FANCA FANCG with FANCA and FANCG of FANCA and FANCG by of FANCA of the of FANCG and the FANCG only in the of FANCA and FANCA that FANCA is of FANCG in cells J. to an in with FANCA multimers to a following with of that multimerization of FANCA intermolecular disulfide FANCA and FANCG in cells exist as monomers under but are to of under of FANCA and FANCG in cells under cells with and of and by an and and of FANCA multimers a in the of cells with to the of of by and an of multimers of proteins a in the of cells with FANCA and FANCG. at in the of to and used as an FANCA and FANCG in of with C a of FANCA and FANCG in cells are to MMC, a DNA-damaging agent that induces DNA damage but also oxidative damage as as of DNA and H. M. treatment also induces a multimeric complex of FANC proteins in cells FANCA and FANCG with H2O2 and multimerization of FANC to H2O2 cells with multimerization of FANCA FANCA multimers to monomers the to in the of of FANCG also multimerization of FANCG in response to treatment that multimerization of FANCA and FANCG is by cells with H2O2 a DNA-damaging agent a multimeric complex of FANCA and FANCG in cells with and with an of H2O2 the and multimerization of FANCA FANCG by and the to in the and used as an in to H2O2 the multimerization of FANCA and we also interaction between FANCA and FANCG in cells with and with cells with H2O2 multimerization of FANCA as as interaction between FANCA and the by a of FANCG with FANCA in a between FANCA and FANCG in vivo by H2O2 treatment. cells with and with an to the with and in the of FANCA and FANCG by and and and the with cells with and with H2O2 with an and with to the to the and and the and and to the and by and an the to the to to treatment of cells multimerization of FANC proteins we also treatment the interaction cells and with H2O2 and of FANCG following of an of with following treatment of cells with H2O2 not of the FANCG with FANCA in a multimerized form of FANCG that at in in the of and also that treatment abolishes multimerization and interaction of FANCA and FANCG in not our results that the interaction may intermolecular disulfide in a oxidative between FANCA and FANCG is in vivo treatment with H2O2 cells with and and the cells and with H2O2 with with an to of and the by and an and an with and to an that not the form of cells exhibit a in a of chromosomal and hypersensitivity to DNA-cross-linking FANC cells and defective in forming the FANC complex J. J. van Weel Arwert F. H. H. and FANCG is of the FANCA and proteins J. J. van Weel Arwert F. H. H. that FANC proteins form a complex that may be their function in damage and repair. we the interaction of FANC proteins in response to oxidative that oxidative DNA multimerization and interaction of FANC proteins that with of at the of the in FANCA and FANCG of and they not have such as FANC proteins multimers under oxidizing in and that the of the FANC complex is via intermolecular disulfide between FANC and proteins are by at disulfide M. J. Full Text Full Text PDF the the of function the of is also to intermolecular disulfide a via disulfide and is an oxidative H. H. H. and complex H. J. Full Text Full Text PDF are the that form their function in response to oxidative be that of the complexes disulfide linkage(s), such as may not be by the complexes are to treatment with M. H. J. the that interaction of FANCA with the interaction is by intermolecular disulfide of FANCA and FANCG be to the of the complex in damage and DNA of FANC proteins the interaction is by H2O2 treatment. H2O2 not only but also DNA to chromosomal and the DNA is by M. MMC, induces and DNA oxidative damage to to interaction between FANCA and FANCG in the F. H. H. J. Full Text Full Text PDF of is of sensitivity in the that sensitivity may also be to a oxidative damage H. is not multimerization of FANC proteins the interaction is to oxidative DNA is a response to DNA-damaging damage not only the interaction but also induces multimerization of FANC proteins (FANCA, and disulfide and not we not the FANCA proteins and of the complex also form multimers under conditions, a that the FANC complex may disulfide following of FANC multimers in response to oxidative is not an that FANCA is of the FANC complex Buchwald M. is that multimers of FANCG with the FANCG with FANCA following oxidative damage multimerization of the FANC proteins a that is the of the FANC interaction is not only crucial the of FANCG J. but also is their function in the interaction with of FANCD2 following oxidative damage not that treatment the FANC proteins in the nuclear and in cells and FANCD2 cells but not in FANCA cells F. J. Full Text Full Text PDF FANCA with cells a of M. van Oostra A.B. H. W. Genet. with is a of FANCD2 M. van Oostra A.B. H. W. Genet. FANCA may an in the of FANCD2 via of and in the Both FANCA and FANCD2 are and their to be by DNA damage H. Full Text Full Text PDF be to the FANCA also interaction between FANCA and and forming a as a to the of a FANC complex at least five FANC proteins (FANCA, -C, -E, -F, and -G) following oxidative damage FANC complex as M. van Oostra A.B. H. W. Genet. a of FANC and of FANCD2 to nuclear following DNA damage M. van Oostra A.B. H. W. Genet. FANC complex also with a in a of F. H. M. M. M. C.G. Hum. Genet. the FANC complex is with the a complex and M. H. W. be to the interaction via disulfide is of the FANC complex in the and DNA the of the FANC complex in response to oxidative stress/damage. Fanconi anemia (FANC) 1The abbreviations used are: FANC, Fanconi anemia; MMC, mitomycin C; DTT, dithiothreitol; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase.1The abbreviations used are: FANC, Fanconi anemia; MMC, mitomycin C; DTT, dithiothreitol; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase. is an autosomal recessive disorder characterized by chromosomal instability and defective DNA repair, and FANC-deficient cells exhibit extreme sensitivity toward oxygen and DNA-cross-linking agents such as diepoxybutane and mitomycin C (1Joenje H. Oostra A.B. Wijker M. di Summa F.M. van Berkel C.G. Rooimans M.A. Ebell W. van Weel M. Pronk J.C. Buchwald M. Arwert F. Am. J. Hum. Genet. 1997; 61: 940-944Abstract Full Text PDF H. Genet. M. H. H. of of FANC have been identified and (FANCA, and (1Joenje H. Oostra A.B. Wijker M. di Summa F.M. van Berkel C.G. Rooimans M.A. Ebell W. van Weel M. Pronk J.C. Buchwald M. Arwert F. Am. J. Hum. Genet. 1997; 61: 940-944Abstract Full Text PDF H. Genet. M. H. H. in of the genes lead to FANC a of genetic with that of DNA that a FANCA and FANCG proteins are part of a nuclear complex their and the of complex results in the and to FANC J. FANCA a and with in the H. FANCA cells a FANC defective in their and to to a FANCA to the mitomycin C sensitivity of that FANCA may be in nuclear of function in repair. FANCG a that been identified as the that is to agents, and DNA-cross-linking agents M. van 1997; cells also show a of chromosomal that be by of 1997; of the of FANC proteins in DNA by the interaction of with following DNA damage J. M. Full Text Full Text PDF is to and is FANC genes in that is the of in response to F. H. that may be in repair. FANC a following H2O2 a FANC proteins in and of oxidative DNA J. H. Buchwald M. M. J. H. J. Full Text Full Text PDF and not between FANCA and FANCG by and J. J. van Weel Arwert F. H. H. F. H. H. J. Full Text Full Text PDF H. H. C.G. Buchwald M. the of FANC proteins have to be is a the FANC proteins in DNA and damage we the interaction of FANC proteins following oxidative that FANCA and FANCG are multimerized and/or form a complex via intermolecular disulfide in response to oxidative as as interaction with of FANCD2 H2O2 that interaction may be an response to oxidative damage that a crucial in DNA and damage and and in with FANCA and and FANCG and and and and and H2O2 of FANCA and FANCG FANCG by the and the Both a and the also a and by DNA FANCA at and by the and by and DNA cells FANCG at by the and the by and DNA and of cells and cells in and which with and in cells in with H2O2 at the at cells with and the by of in in the of and to and with a at and the with and with and proteins by used in to the at at least with that been in with in the the with of proteins in cells following J. and by and by a M. J. 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M. treatment also induces a multimeric complex of FANC proteins in cells FANCA and FANCG with H2O2 and multimerization of FANC to H2O2 cells with multimerization of FANCA FANCA multimers to monomers the to in the of of FANCG also multimerization of FANCG in response to treatment that multimerization of FANCA and FANCG is by cells with H2O2 a DNA-damaging agent a multimeric complex of FANCA and FANCG in cells with and with an of H2O2 the and multimerization of FANCA FANCG by and the to in the and used as an in to H2O2 the multimerization of FANCA and we also interaction between FANCA and FANCG in cells with and with cells with H2O2 multimerization of FANCA as as interaction between FANCA and the by a of FANCG with FANCA in a between FANCA and FANCG in vivo by H2O2 treatment. cells with and with an to the with and in the of FANCA and FANCG by and and and the with cells with and with H2O2 with an and with to the to the and and the and and to the and by and an the to the to to treatment of cells multimerization of FANC proteins we also treatment the interaction cells and with H2O2 and of FANCG following of an of with following treatment of cells with H2O2 not of the FANCG with FANCA in a multimerized form of FANCG that at in in the of and also that treatment abolishes multimerization and interaction of FANCA and FANCG in not our results that the interaction may intermolecular disulfide in a oxidative between FANCA and FANCG is in vivo treatment with H2O2 cells with and and the cells and with H2O2 with with an to of and the by and an and an with and to an that not the form of of FANC under to a FANC proteins in and the of oxidative H. with cells FANC genes hypersensitivity to H2O2 treatment H. J. Full Text Full Text PDF and not FANC proteins (FANCA, and of we of in of FANC cells with FANCG by following a and the of as multimers in under oxidizing conditions, whereas as a with of to the of intermolecular disulfide FANCG but not the of the not form multimers of is to that form of multimers in a to a under oxidizing and of of FANCA and FANCG in a of FANC cells with a FANCA FANCG with FANCA and FANCG of FANCA and FANCG by of FANCA of the of FANCG and the FANCG only in the of FANCA and FANCA that FANCA is of FANCG in cells J. to an in with FANCA multimers to a following with of that multimerization of FANCA intermolecular disulfide FANCA and FANCG in cells exist as monomers under but are to of under Treatment of with C a of FANCA and FANCG in cells are to MMC, a DNA-damaging agent that induces DNA damage but also oxidative damage as as of DNA and H. M. treatment also induces a multimeric complex of FANC proteins in cells FANCA and FANCG with H2O2 and multimerization of FANC to H2O2 cells with multimerization of FANCA FANCA multimers to monomers the to in the of of FANCG also multimerization of FANCG in response to treatment that multimerization of FANCA and FANCG is by in to H2O2 the multimerization of FANCA and we also interaction between FANCA and FANCG in cells with and with cells with H2O2 multimerization of FANCA as as interaction between FANCA and the by a of FANCG with FANCA in a treatment of cells multimerization of FANC proteins we also treatment the interaction cells and with H2O2 and of FANCG following of an of with following treatment of cells with H2O2 not of the FANCG with FANCA in a multimerized form of FANCG that at in in the of and also that treatment abolishes multimerization and interaction of FANCA and FANCG in not our results that the interaction may intermolecular disulfide in a oxidative cells exhibit a in a of chromosomal and hypersensitivity to DNA-cross-linking FANC cells and defective in forming the FANC complex J. J. van Weel Arwert F. H. H. and FANCG is of the FANCA and proteins J. J. van Weel Arwert F. H. H. that FANC proteins form a complex that may be their function in damage and repair. we the interaction of FANC proteins in response to oxidative that oxidative DNA multimerization and interaction of FANC proteins that with of at the of the in FANCA and FANCG of and they not have such as FANC proteins multimers under oxidizing in and that the of the FANC complex is via intermolecular disulfide between FANC and proteins are by at disulfide M. J. Full Text Full Text PDF the the of function the of is also to intermolecular disulfide a via disulfide and is an oxidative H. H. H. and complex H. J. Full Text Full Text PDF are the that form their function in response to oxidative be that of the complexes disulfide linkage(s), such as may not be by the complexes are to treatment with M. H. J. the that interaction of FANCA with the interaction is by intermolecular disulfide of FANCA and FANCG be to the of the complex in damage and DNA of FANC proteins the interaction is by H2O2 treatment. H2O2 not only but also DNA to chromosomal and the DNA is by M. MMC, induces and DNA oxidative damage to to interaction between FANCA and FANCG in the F. H. H. J. Full Text Full Text PDF of is of sensitivity in the that sensitivity may also be to a oxidative damage H. is not multimerization of FANC proteins the interaction is to oxidative DNA is a response to DNA-damaging damage not only the interaction but also induces multimerization of FANC proteins (FANCA, and disulfide and not we not the FANCA proteins and of the complex also form multimers under conditions, a that the FANC complex may disulfide following of FANC multimers in response to oxidative is not an that FANCA is of the FANC complex Buchwald M. is that multimers of FANCG with the FANCG with FANCA following oxidative damage multimerization of the FANC proteins a that is the of the FANC interaction is not only crucial the of FANCG J. but also is their function in the interaction with of FANCD2 following oxidative damage not that treatment the FANC proteins in the nuclear and in cells and FANCD2 cells but not in FANCA cells F. J. Full Text Full Text PDF FANCA with cells a of M. van Oostra A.B. H. W. Genet. with is a of FANCD2 M. van Oostra A.B. H. W. Genet. FANCA may an in the of FANCD2 via of and in the Both FANCA and FANCD2 are and their to be by DNA damage H. Full Text Full Text PDF be to the FANCA also interaction between FANCA and and forming a as a to the of a FANC complex at least five FANC proteins (FANCA, -C, -E, -F, and -G) following oxidative damage FANC complex as M. van Oostra A.B. H. W. Genet. a of FANC and of FANCD2 to nuclear following DNA damage M. van Oostra A.B. H. W. Genet. FANC complex also with a in a of F. H. M. M. M. C.G. Hum. Genet. the FANC complex is with the a complex and M. H. W. be to the interaction via disulfide is of the FANC complex in the and DNA FANC cells exhibit a in a of chromosomal and hypersensitivity to DNA-cross-linking FANC cells and defective in forming the FANC complex J. J. van Weel Arwert F. H. H. and FANCG is of the FANCA and proteins J. J. van Weel Arwert F. H. H. that FANC proteins form a complex that may be their function in damage and repair. we the interaction of FANC proteins in response to oxidative that oxidative DNA multimerization and interaction of FANC proteins that with of at the of the in FANCA and FANCG of and they not have such as FANC proteins multimers under oxidizing in and that the of the FANC complex is via intermolecular disulfide between FANC and proteins are by at disulfide M. J. Full Text Full Text PDF the the of function the of is also to intermolecular disulfide a via disulfide and is an oxidative H. H. H. and complex H. J. Full Text Full Text PDF are the that form their function in response to oxidative be that of the complexes disulfide linkage(s), such as may not be by the complexes are to treatment with M. H. J. the that interaction of FANCA with the interaction is by intermolecular disulfide of FANCA and FANCG be to the of the complex in damage and DNA repair. of FANC proteins the interaction is by H2O2 treatment. H2O2 not only but also DNA to chromosomal and the DNA is by M. MMC, induces and DNA oxidative damage to to interaction between FANCA and FANCG in the F. H. H. J. Full Text Full Text PDF of is of sensitivity in the that sensitivity may also be to a oxidative damage H. is not multimerization of FANC proteins the interaction is to oxidative DNA is a response to DNA-damaging damage not only the interaction but also induces multimerization of FANC proteins (FANCA, and disulfide and not we not the FANCA proteins and of the complex also form multimers under conditions, a that the FANC complex may disulfide following of FANC multimers in response to oxidative is not an that FANCA is of the FANC complex Buchwald M. is that multimers of FANCG with the FANCG with FANCA following oxidative damage multimerization of the FANC proteins a that is the of the FANC interaction is not only crucial the of FANCG J. but also is their function in the interaction with of FANCD2 following oxidative damage not that treatment the FANC proteins in the nuclear and in cells and FANCD2 cells but not in FANCA cells F. J. Full Text Full Text PDF FANCA with cells a of M. van Oostra A.B. H. W. Genet. with is a of FANCD2 M. van Oostra A.B. H. W. Genet. FANCA may an in the of FANCD2 via of and in the Both FANCA and FANCD2 are and their to be by DNA damage H. Full Text Full Text PDF be to the FANCA also interaction between FANCA and FANCG. FANCA and forming a as a to the of a FANC complex at least five FANC proteins (FANCA, -C, -E, -F, and -G) following oxidative damage FANC complex as M. van Oostra A.B. H. W. Genet. a of FANC and of FANCD2 to nuclear following DNA damage M. van Oostra A.B. H. W. Genet. FANC complex also with a in a of F. H. M. M. M. C.G. Hum. Genet. the FANC complex is with the a complex and M. H. W. be to the interaction via disulfide is of the FANC complex in the and DNA the of