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.

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 mustard-hypersensitive 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 BRCA1, is involved in maintaining genomic stability in response not only to DNA interstrand crosslinks but also a range of other DNA damages including DNA strand breaks. NM3 and other "FA-like" Chinese hamster mutants should provide an important resource for the study of these processes in mammalian cells.

[1]  D. Papadopoulo,et al.  Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia. , 1998, Journal of molecular biology.

[2]  P. Telleman,et al.  Genetic diversity of mitomycin C-hypersensitive Chinese hamster cell mutants: A new complementation group with chromosomal instability , 1993, Somatic cell and molecular genetics.

[3]  D. Näf,et al.  Fanconi Anemia Proteins FANCA, FANCC, and FANCG/XRCC9 Interact in a Functional Nuclear Complex , 1999, Molecular and Cellular Biology.

[4]  P. Jeggo,et al.  DNA double-strand break repair pathways and cellular tolerance to inhibitors of topoisomerase II. , 1990, Cancer Research.

[5]  M. Buchwald,et al.  Evidence for at least eight Fanconi anemia genes. , 1997, American journal of human genetics.

[6]  P. Bryant,et al.  Isolation of camptothecin-sensitive chinese hamster cell mutants: phenotypic heterogeneity within the ataxia telangiectasia-like XRCC8 (irs2) complementation group. , 2000, Mutagenesis.

[7]  M. Buchwald,et al.  Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents. , 1982, Cancer research.

[8]  A. Carrano,et al.  Cytogenetic characterization of the ionizing radiation-sensitive Chinese hamster mutant irs1. , 1991, Mutation research.

[9]  M. Tomasz,et al.  Mitomycin C: small, fast and deadly (but very selective). , 1995, Chemistry & biology.

[10]  C. Arlett,et al.  The response of a variety of human fibroblast cell strains to the lethal effects of alkylating agents. , 1982, Carcinogenesis.

[11]  A. D’Andrea,et al.  Molecular biology of Fanconi anemia: implications for diagnosis and therapy. , 1997, Blood.

[12]  F. Arwert,et al.  The Chinese hamster cell mutant V-H4 is homologous to Fanconi anemia (complementation group A). , 1991, Cytogenetics and cell genetics.

[13]  J. Thacker,et al.  Six complementation groups for ionising-radiation sensitivity in Chinese hamster cells. , 1988, Mutation research.

[14]  C. Mathew,et al.  Complementation analysis in Fanconi anemia: assignment of the reference FA-H patient to group A. , 2000, American journal of human genetics.

[15]  A. D’Andrea,et al.  The fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  C G Mathew,et al.  Isolation of a cDNA representing the Fanconi anemia complementation group E gene. , 2000, American journal of human genetics.

[17]  C. Arlett,et al.  Survey of radiosensitivity in a variety of human cell strains. , 1980, Cancer research.

[18]  L. Povirk,et al.  DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes. , 1996, Mutation research.

[19]  J. Albala,et al.  The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. D’Andrea,et al.  Carboxy terminal region of the Fanconi anemia protein, FANCG/XRCC9, is required for functional activity. , 2000, Blood.

[21]  Hans Joenje,et al.  Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA , 1996, Nature Genetics.

[22]  L. Thompson,et al.  A CHO mutant, UV40, that is sensitive to diverse mutagens and represents a new complementation group of mitomycin C sensitivity. , 1996, Mutation research.

[23]  N. Alon,et al.  Drug sensitivity spectra in Fanconi anemia lymphoblastoid cell lines of defined complementation groups. , 1999, Mutation research.

[24]  M. Jasin,et al.  Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination , 1999, Nature.

[25]  D. Ross,et al.  Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin. , 1996, Biochemical pharmacology.

[26]  C. Walsh,et al.  Phenotypic correction of Fanconi anemia group C knockout mice. , 2000, Blood.

[27]  Q. Waisfisz,et al.  The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG. , 2000, Human molecular genetics.

[28]  D. Murray,et al.  Isolation and characterization of nitrogen mustard-sensitive mutants of Chinese hamster ovary cells. , 1991, Mutation research.

[29]  A. Taylor,et al.  Effects of ionizing radiation on cells from Fanconi's anemia patients. , 1985, Cancer research.

[30]  S. Kajigaya,et al.  Fanconi anemia protein, FANCG, is a phosphoprotein and is upregulated with FANCA after TNF-alpha treatment. , 2001, Biochemical and biophysical research communications.

[31]  R. Weichselbaum,et al.  Response of human fibroblasts to low dose rate gamma irradiation. , 1984, Radiation research.

[32]  A. D’Andrea,et al.  The fanconi anemia proteins FANCA and FANCG stabilize each other and promote the nuclear accumulation of the Fanconi anemia complex. , 2000, Blood.

[33]  W. R. Shannon,et al.  Cloning of cDNAs for Fanconi's anaemia by functional complementation , 1992, Nature.

[34]  B. Alter.  FANCONI'S ANAEMIA AND ITS VARIABILITY , 1994, British journal of haematology.

[35]  L. Thompson,et al.  Biochemical and genetic analysis of the Chinese hamster mutants irs1 and irs2 and their comparison to cultured ataxia telangiectasia cells. , 1990, Mutagenesis.

[36]  K. Brookman,et al.  A screening method for isolating DNA repair-deficient mutants of CHO cells , 1980, Somatic cell genetics.

[37]  S. Ellard,et al.  Cellular and chromosomal hypersensitivity to DNA crosslinking agents and topoisomerase inhibitors in the radiosensitive Chinese hamster irs mutants: phenotypic similarities to ataxia telangiectasia and Fanconi's anaemia cells. , 1993, Carcinogenesis.

[38]  A. Auerbach.  Fanconi anemia diagnosis and the diepoxybutane (DEB) test. , 1993, Experimental hematology.

[39]  N. J. Jones.  Genetic analysis of mitomycin C-hypersensitive Chinese hamster cell mutants. , 1994, Mutagenesis.

[40]  C. Mathew,et al.  Positional cloning of the Fanconi anaemia group A gene , 1996, Nature Genetics.

[41]  D. Busch,et al.  Phenotype of FAECB (Facility for Automated Experiments in Cell Biology) Chinese hamster ovary mutants with minimal UV-sensitivity. , 2001, Mutation research.

[42]  L. Thompson.  Chinese hamster cells meet DNA repair: an entirely acceptable affair. , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[43]  J. Thacker,et al.  DNA-break repair, radioresistance of DNA synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: comparisons to ataxia-telangiectasia cells. , 1990, Mutation research.

[44]  J. Thacker,et al.  Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells. , 1987, Mutation research.

[45]  S. Ganesan,et al.  Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. , 2001, Molecular cell.

[46]  S. Wolff,et al.  New Giemsa method for the differential staining of sister chromatids , 1974, Nature.

[47]  Zdzienicka Mz.  Mammalian X ray sensitive mutants: a tool for the elucidation of the cellular response to ionizing radiation. , 1996 .

[48]  B. Alter.  Fanconi's anemia and malignancies , 1996, American journal of hematology.

[49]  Hans Joenje,et al.  The Fanconi anaemia group G gene FANCG is identical with XRCC9 , 1998, Nature Genetics.

[50]  J. Lamerdin,et al.  XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. , 1998, Molecular cell.

[51]  Hans Joenje,et al.  The Fanconi anaemia gene FANCF encodes a novel protein with homology to ROM , 2000, Nature Genetics.

[52]  C. Mathew,et al.  Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9 , 2000, European Journal of Human Genetics.

[53]  Q. Waisfisz,et al.  A physical complex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[54]  C. Lewis,et al.  Association of complementation group and mutation type with clinical outcome in fanconi anemia. European Fanconi Anemia Research Group. , 2000, Blood.

[55]  J. Thacker.  Resistance to methyl mercaptopurine riboside in cultured hamster cells. Preliminary characterization of resistant cells and conditions affecting their selection in quantitative mutation studies. , 1980, Mutation research.

[56]  R. Moses,et al.  Positional cloning of a novel Fanconi anemia gene, FANCD2. , 2001, Molecular cell.

[57]  M. Waters,et al.  A review of the genetic and related effects of 1,3-butadiene in rodents and humans. , 2000, Mutation research.

[58]  R. Tebbs,et al.  Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[59]  J. Thacker,et al.  The genetic basis of resistance to ionising radiation damage in cultured mammalian cells. , 1991, Mutation research.

[60]  C. Mathew,et al.  Direct interactions of the five known Fanconi anaemia proteins suggest a common functional pathway. , 2001, Human molecular genetics.

[61]  P. Telleman,et al.  A new complementation group of mitomycin C-hypersensitive Chinese hamster cell mutants that closely resembles the phenotype of fanconi anemia cells. , 1995, Cancer research.

[62]  D. Näf,et al.  Molecular biology of Fanconi anemia. , 1997, Hematology/oncology clinics of North America.

[63]  H. Strutt,et al.  Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double strand breaks in replicating DNA. , 1991, Nucleic acids research.

[64]  N. Alon,et al.  Erratum: Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA , 1996, Nature Genetics.

[65]  N. J. Jones,et al.  The isolation and genetic analysis of V79-derived etoposide sensitive Chinese hamster cell mutants: two new complementation groups of etoposide sensitive mutants. , 1999, Mutation research.