|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
PLENARY PAPER
From the Department of Pediatric Oncology, Dana-Farber
Cancer Institute, and Department of Pediatrics, Children's Hospital,
Harvard Medical School, Boston, MA; and Institut fur Humangenetik,
Charite-Campus Virchow-Klinikum, Humbodt Universitat Berlin, Germany.
Fanconi anemia (FA) is an autosomal recessive cancer susceptibility
syndrome with eight complementation groups. Four of the FA genes have
been cloned, and at least three of the encoded proteins, FANCA, FANCC,
and FANCG/XRCC9, interact in a nuclear complex, required for the
maintenance of normal chromosome stability. In the current study,
mutant forms of the FANCA and FANCG proteins have been generated and
analyzed with respect to protein complex formation, nuclear
translocation, and functional activity. The results demonstrate that
the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds
to the amino terminal nuclear localization signal (NLS) of the FANCA
protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is
a direct protein-protein interaction. Interestingly, a truncated mutant
form of the FANCG protein, lacking the carboxy terminus, binds in a
complex with FANCA and translocates to the nucleus; however, this
mutant protein fails to bind to FANCC and fails to correct the
mitomycin C sensitivity of an FA-G cell line. Taken together, these
results demonstrate that binding of FANCG to the amino terminal FANCA
NLS sequence is necessary but not sufficient for the functional
activity of FANCG. Additional amino acid sequences at the carboxy
terminus of FANCG are required for the binding of FANCC in the complex.
(Blood. 2000;96:1625-1632) Fanconi anemia (FA) is an autosomal recessive
cancer susceptibility syndrome characterized by multiple congenital
anomalies, progressive bone marrow failure, and cellular sensitivity to
DNA cross-linking agents (reviewed in Auerbach et al1 and
Garcia-Higuera et al2). According to somatic cell fusion
studies, FA is comprised of 8 distinct complementation
groups.3 Four of the FA genes, including the FANCA, FANCG,
FANCC, and FANCF genes, have been cloned.4-8 The FANCG
gene is identical to the XRCC9 gene.9 Two additional
genes, for FANCD10 and FANCE,11 have been
mapped. The 4 cloned FA proteins have little or no homology to each
other or to other proteins in the database, and little is known
regarding their cellular function. On the basis of the similar clinical and cellular phenotypes observed among the 8 FA complementation groups,
the FA proteins appear to cooperate in a common cellular pathway.
Cells derived from patients with FA display a broad range of
abnormalities (reviewed in Liu et al12 and D'Andrea and
Grompe13). FA cells are sensitive to DNA cross-linking
agents and oxygen radicals14,15 and have spontaneous
chromosome breakage.16 FA cells also have cell cycle
abnormalities and exhibit a prolongation in the G2 phase of the cell
cycle.17,18 FA cells have increased cellular sensitivity
to the apoptotic effects of tumor necrosis factor Increasing evidence demonstrates that the FA proteins cooperate in a
novel cellular pathway.23 Three of the cloned proteins, FANCA, FANCG, and FANCC, bind and interact in a protein
complex.24-26 Interestingly, this protein complex is not
observed in FA cells derived from other FA complementation groups,
including groups B, E, F, and H.27 These results suggest
that the products of other FA genes, such as the recently cloned FANCF
protein,8 regulate the formation of the complex, perhaps
by serving as other adaptor proteins or enzymes regulating protein
complex assembly. The FA-D complementation group is distinct from the
other groups. The FA protein complex forms normally in FA-D cells,
suggesting that the product of the FANCD gene functions downstream of
complex formation.
Patient-derived mutant forms of FA proteins have been instructive in
delineating functional domains of these proteins and in defining the
biochemical features of the FA pathway. Point mutations in the
carboxy-terminus of FANCC, such as the patient-derived FANCC-L554P
mutation,5 block complex formation.28 Also,
point mutations in the carboxy terminus of FANCA, especially in the region of the partial leucine zipper, disrupt the
complex.29 Point mutations, such as the FANCA(H1110P), are
defective in FANCC binding, FANCA phosphorylation, and nuclear
localization, thereby defining many of the normal biochemical events in
the FA pathway.27
In the current study, we performed a detailed structure/function
analysis of FANCA and FANCG to determine the regions of the proteins
required for assembly and for nuclear accumulation. On the basis of
this analysis, the amino terminal two-thirds of FANCG binds directly to
an amino-terminal domain of FANCA containing the nuclear localization
signal (NLS) region. Interestingly, an FA patient-derived truncated
mutant form of FANCG, lacking the carboxy terminal 39 amino acids,
binds to FANCA but fails to recruit FANCC to the complex and fails to
complement an FA-G cell line. This result suggests that the 39 amino
acids at the carboxyl terminus of FANCG are required for both
functional activity and FANCC binding in the complex.
Cell culture
Construction of expression vectors encoding FA proteins
For construction of FANCG pMMP-puro mutants, the cDNAs encoding various
truncation mutants of FANCG were amplified by PCR and subcloned into
the retroviral expression vector, pMMP-puro.31 The
FANCG(Q26R), FANCG For construction of FANCG and FANCA fusion proteins in yeast 2-hybrid interaction studies, the cDNAs encoding full-length FANCG or FANCG (amino acid 134-278) were subcloned into the EcoRI and BamHI sites of the vector, pAS2-1 (Clontech, Palo Alto, CA). The cDNA encoding FANCA (amino acid 1-230) was subcloned into the BamHI and Xho sites of pACT2 vector (Clontech). The cDNA inserts were verified by DNA sequencing. Cotransfection of FANCA and FANCG cDNAs in COS cells For protein expression screening, COS cells were transfected as previously described.32 Forty-eight hours after transfection, cells were directly solubilized in SDS sample buffer, boiled, and loaded to SDS/PAGE. Western blots were performed by using a monoclonal antibody against HA (monoclonal #11) (Bibco Inc, Berkeley, CA).For immunoprecipitation experiments, COS cells were plated onto 10-cm plates to achieve 40% confluence on the day before transfection. Transfection was performed by using maxiprep plasmid DNAs (5 µg/plate) and 40 µL of lipofectamine (Life Technologies, Gaithersburg, MD) per plate. Two days after transfection, cells were scraped in cold phosphate-buffered saline and pelleted. Immunoprecipitation was performed as previously described.24 35S-labeled in vitro translation FANCG and FANCA mutant cDNAs, subcloned into the pcDNA vector, were translated with amino acid mix together with 35S-methionine (1000 Ci/mmol; Amersham Pharmarcia, Piscataway, NJ) according to manufacturer's specifications (TNT Lysate Coupled Transcription/Translation kit, Promega, Madison, WI). For FANCG and FANCA binding experiments, the in vitro-translated protein products were mixed for 30 minutes at 30°C in reaction buffer, 50 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.5% NP-40, 1% Triton, 3% glycerol, pH 7.4, followed by 30 minutes on ice. The reactions were diluted with the same reaction buffer, and immunoprecipitations were performed. The samples were subjected to SDS-PAGE, and the dried gel was exposed to x-ray films (Kodak, Rochester, NY).FANCA-NLS peptide competition assay Two peptides were synthesized for the assay. The sequence of FANCA-NLS-wt was GRRRAWAELLAGRVKREKYN, and the sequence of FANCA-NLS-mut2 was GNNNAWAELLAGNVIDEQYN.30 In vitro-translated FANCG protein was preincubated with either NLS-wt or NLS-mut2 peptides for 30 minutes at 30°C in the reaction buffer described in the previous experiment, before mixing with in vitro-translated HA-FANCA (amino acid 1-240) protein. The concentrations of the peptides are indicated in the figure legends. Immunoprecipitation and SDS-PAGE were performed as previously described.Interaction of FANCG and FANCA in yeast FANCG/pAS2-1 and FANCA-NLS/pACT2 were cotransformed into yeast strain Y190 and grown on selective media (-Trp, -Leu). Yeast cells were lysed, according to standard protocol (Clontech). The cell lysate samples were subject to SDS-PAGE. The expression of both FANCG and FANCA proteins were confirmed by Western blots with antibodies against the GAL4 DNA-binding domain and activation domain (Santa Cruz, Santa Cruz, CA), respectively. Clones that expressed both proteins were tested for their ability to activate GAL4 transcription by colony-lift filter assay with proper controls.Retroviral infection of FA cell lines The indicated pMMP constructs were transfected by lipofection into 293 producer cells (human embryonic kidney cells) expressing the VSV-G envelope protein.33 Retroviral supernatants were collected on day 5 following lipofection and contained 4.6 × 106 infectious units/mL, as estimated by Southern blot analysis of infected NIH-3T3 cells (data not shown).FA lymphoblasts were infected with the various pMMP supernatants by a 6-hour incubation in the presence of 8 µg/mL polybrene. Infected cells were washed free of viral supernatant and resuspended in growth media. After 48 hours, cells were transferred to media containing puromycin (1 µg/mL). Dead cells were removed over a Ficoll cushion after 5 days, and surviving cells were grown under continuous selection in puromycin. FA fibroblast lines were retrovirally transduced as previously described.30 Mitomycin C assay Mitomycin C (MMC) sensitivity assays for lymphoblasts were performed as previously described.30Immunofluorescence microscopy Immunofluorescence microscopy of human fibroblasts was performed as previously described.30
The amino terminal region of FANCA binds to FANCG. Recent
studies30,34 have shown that the amino terminal region of
FANCA is required for FANCC coimmunoprecipitation, nuclear
localization, and functional complementation of an FA-A cell line. This
amino terminal region of FANCA contains a functional bipartite NLS. To
further assess the importance of the amino terminal region of FANCA in
FANCG binding, we generated various FANCA mutants (Figure
1A). Initially, we cotransfected COS
cells with the cDNAs encoding these HA-epitope tagged FANCA mutant
proteins along with the wild-type FANCG cDNA (Figure 1B). We used an
anti-FANCG antiserum to immunoprecipitate FANCG and FANCG binding
proteins. As previously described, the full-length FANCA protein
coimmunoprecipitated with FANCG (data not shown). A truncated mutant
form of FANCA (FANCA 1-240), containing the amino terminal 240 amino
acids of FANCA, was also coimmunoprecipitated with FANCG (upper blot,
lane 5). In contrast, a truncated mutant form of FANCA (FANCA
722-1455), containing the carboxy terminal 723 amino acids of FANCA,
failed to coimmunoprecipitate with FANCG (upper blot, lane 6), even
though the FANCA 722-1455 truncated mutant was well expressed (lower blot, lane 6). These differential binding data (summarized in Figure
1A) demonstrate that the amino terminal 240 amino acids of FANCA are
necessary and sufficient for FANCG binding.
Amino terminal two-thirds of FANCG binds to FANCA To determine the region of FANCG required for FANCA binding, we next generated a large series of cDNAs encoding mutant FANCG proteins (Figure 2A). One of these FANCG mutant proteins (FANCG1749 A) was originally identified by mutational
screening of a known FA-G patient. Another variant FANCG protein
(FANCGexon13) is modeled from a splice variant of the FANCG gene
found in normal human lymphoblasts, resulting from skipping of exon 13 (Y. Kuang, unpublished observation). We cotransfected COS cells with a
subset of cDNAs encoding HA-epitope tagged FANCG mutant proteins along
with the wild-type FANCA cDNA (Figure 2B). We used an anti-FANCA
antiserum to immunoprecipitate FANCA and FANCG proteins. The
full-length FANCG protein coimmunoprecipitated with FANCA (Figure 2B,
lanes 3, 12, and 18). Interestingly, a truncated mutant form of FANCG, containing the amino terminal 428 amino acids of FANCG,
coimmunoprecipitated with FANCA (Figure 2B, lane 19). Further
truncations of the amino terminal region of FANCG resulted in loss of
FANCA binding (lanes 4, 5, and 13). These binding data (summarized in
Figure 2A) demonstrate that the amino terminal 428 amino acids of FANCG
are necessary and sufficient for FANCA binding.
FANCG binds directly to the NLS of FANCA Several groups,24,35 including our own, have used in vitro-translated proteins to demonstrate that FANCA binds to FANCG. To further investigate whether the interaction of FANCA and FANCG is truly a direct protein-protein interaction, we used the yeast 2-hybrid method (Figure 3).36-39 The amino terminal 230 amino acids of FANCA were translationally fused to the activation domain of GAL4 in the pACT2 expression vector (Figure 3A). In addition, variable regions of the FANCG protein were translationally fused to the DNA-binding domain in the pAS2-1 expression vector. The various FANCA and FANCG proteins were coexpressed in Y190 cells. A strong interaction between the amino terminus of FANCA and FANCG was observed, based on the generation of blue colonies in the colony-lift filter assay (Figure 3B). These data further demonstrate that the amino terminus of FANCA interacts with the amino terminus of FANCG and that the interaction is direct.
To further implicate the NLS region of FANCA as the direct binding site
of FANCG, we next performed the coimmunoprecipitation of in
vitro-translated FANCA 1-240 and FANCG in the presence of a specific
peptide inhibitor (Figure 4).
Interestingly, a 20 amino acid peptide containing the NLS region of
FANCA30 specifically competed the coimmunoprecipitation of
FANCA 1-240 and FANCG. A mutant peptide, in which all basic amino acids
of the NLS have been mutated, failed to compete the binding of FANCA
and FANCG. Our previous studies30 had shown that these
amino acid changes also disrupt the functional activity of the NLS
motif. Densitometric scanning of the autoradiograph in Figure 4A
demonstrated that the inhibition was dependent on the concentration of
free peptide (Figure 4B). One-half maximal inhibition was detected at a
wild-type peptide concentration of approximately 0.9 µmol/L. These
data further confirm that FANCG binds directly to the NLS region of FANCA.
Amino terminal region of FANCG is necessary but not sufficient for functional complementation of an FA-G cell line We next tested mutant forms of the FANCG protein for their ability to functionally complement the MMC sensitivity of an FA-G lymphoblast line, EUFA316. The cDNAs encoding various forms of FANCG were retrovirally transduced into EUFA316 cells, and stably transduced cells were analyzed for MMC sensitivity (Figure 5). As previously shown,4 the wild-type full-length FANCG corrected the MMC sensitivity of FA-G cells (closed circles). Three truncated forms of FANCG (FANCG 1-428, FANCGexon13, and FANCG1749A), failed to complement the FA-G
cells. The failure of the patient-derived FANCG1749A protein to
complement the FA-G cells confirms that this is a bona fide
FANCG mutation.
We next analyzed the ability of the truncated forms of the FANCG
protein to form a complex with the endogenous FANCA and FANCC proteins
in these transfected FA-G lymphoblasts (Figure
6). Whole cell extract Western blotting
(Figure 6, bottom blot) indicated that the three FANCG-truncated
proteins (lanes 2, 3, and 4) were expressed at a similar level,
compared to FANCG(wt) (lane 5). This control experiment suggests that
the failure to complement the FA-G cell line by these mutant FANCG
proteins (Figure 5) did not result from the low level of expression of
these proteins. As previously shown,24
immunoprecipitation of the FANCA protein with an anti-FANCA
antiserum resulted in the coimmunoprecipitation of wild-type FANCC
(lane 5, anti-FANCC immunoblot) and wild-type FANCG (lane 5, anti-FANCG
immunoblot). The mutant FANCG proteins, FANCG 1-428, FANCG
Carboxy terminal truncated mutant forms of FANCG localize to the nucleus As a control, we next examined the cellular localization of the various mutant forms of FANCG by immunofluorescence (Figure 7). Various forms of FANCG were expressed in the FA-G fibroblast line, DF3. These fibroblasts are derived from an FA-G patient, have a homozygous mutation in the FANCG gene (FANCG 1665 G > C), and fail to express endogenous FANCG protein (Y. Kuang, unpublished observation). DF3 fibroblasts were functionally complemented by retroviral transduction with the wild-type FANCG cDNA (data not shown). The wild-type FANCG protein and the mutant FANCG 1-428 protein localized to the nucleus of the transduced DF3 cells similarly (Figure 7). The failure of the FANCG 1-428 protein to complement the cells was, therefore, not due to its failure to localize to the nucleus. In addition, we tested a variant form of FANCG with a Q to R mutation at amino acid 26. This variant allele was found in cells from several FA-G patients and normal control subjects, suggesting that the Q26R change is a polymorphism and is not a pathogenic mutation. Consistent with this hypothesis, FANCG(Q26R) protein binds to FANCA and FANCC and corrects the MMC sensitivity of the transduced FA-G cells (data not shown). Moreover, the FANCG(Q26R) protein accumulates normally in the nucleus of transduced cells (Figure 7).
Several lines of evidence demonstrate that the FA proteins, including FANCA, FANCG, and FANCC, interact in a common cellular pathway, leading to the accumulation of the FA protein complex in the nucleus. Absence of this nuclear protein complex, resulting from biallelic germline mutations of any one of these FA genes, correlates with chromosome instability and a broad array of cellular and clinical abnormalities. The protein products of additional (uncloned) FA genes appear to be required for the assembly, stability, and nuclear transport of the FA complex.27 In the current study, we identified the regions of FANCG and FANCA required for their binding interaction and identified a carboxy terminal domain of FANCG required for its function and for the recruitment of FANCC to the complex. We also identified patient-derived truncated mutant forms of FANCG that coimmunoprecipitated with FANCA but failed to bind FANCC and failed to functionally complement FA-G cells. Several structural features of the interaction between the FANCA and FANCG proteins were identified. First, we have identified the regions of the FANCA and FANCG proteins required for their interaction. The amino terminal two-thirds of FANCG binds to an amino terminal region of FANCA. Second, we used a 2-hybrid analysis to determine that the amino terminus of FANCA binds directly to FANCG. Consistent with these studies, FANCA binds to FANCG even in cells derived from other FA complementation groups.24 These results confirm that the products of other FA genes are not required for the FANCA/FANCG protein interaction. Direct binding between FANCA and FANCG has also been recently reported by another group.40 Third, we have demonstrated that FANCG binds directly to the NLS region
of FANCA. This interaction is competed by a 20 amino acid peptide
corresponding to the amino terminal region of FANCA. Mutations in the
NLS region that disrupt nuclear localization of FANCA30
also disrupt the interaction between FANCA and FANCG. Disruption of the
FA protein complex with this NLS peptide may provide an experimental
strategy for promoting MMC sensitivity in otherwise normal cells. Given
the direct interaction of FANCG with the NLS of FANCA, alternative
models may account for their possible functional interaction in the
process of nuclear uptake. FANCG may cooperate with importin- Fourth, the interaction of FANCA and FANCG is distinct from the interaction of FANCA and FANCC. Although the FANCA/FANCG binding interaction is direct, the FANCA/FANCC and FANCG/FANCC interactions appear to be indirect. FANCA and FANCC interactions appear to be weak, based on binding of in vitro-translated proteins24 or 2-hybrid analysis.40,43 The binding of FANCA and FANCC may, therefore, require additional adaptor proteins or posttranslational modifications. Interestingly, the FANCA/FANCC interaction is not observed in several other FA complementation groups, including groups B, E, F, and H, suggesting that their interaction is regulated by other FA gene products.27 For instance, the recently cloned FANCF protein8 may also be a component of the FA protein complex required for FANCC binding. Accordingly, the purification of the FA protein complex may allow the identification of other FA gene products. Fifth, our data elucidate various structural requirements for the
interaction of FANCC with the FANCA/FANCG complex. Our previous studies30 demonstrated that the amino terminal NLS region
of FANCA is required for FANCC binding. Because the NLS region of FANCA
binds directly to FANCG, FANCA/FANCG binding appears to be required for
FANCC binding in the complex. Also, point mutations in the leucine
zipper region of FANCA disrupt the FANCC interaction, suggesting that
this FANCA region also contributes to FANCC binding. Finally, in the
current study, the three FANCG mutant proteins, FANCG 1-428, FANCG The FANCG1749 It is interesting that mutant forms of FA proteins bind and form complexes with other endogenous wild-type FA proteins. For instance, previous studies24,44 have shown that the FANCA mutant protein, FANCA(H1110P), forms a complex with wild-type FANCG. In the current study, we have shown that the carboxy terminal truncated forms of FANCG bind wild-type FANCA. Whether or not these mutant proteins have dominant negative activity in vitro or account for the phenotypic variation among FA patients remains to be determined. Our observations demonstrate that formation of the FANCA/FANCG protein complex is necessary but not sufficient for functional activity of the FA pathway. Similarly, in FA-D cells the FA protein complex forms normally, yet these cells remain sensitive to MMC.27 Finally, our results confirm and extend the results of Kruyt et
al.45 These investigators determined that the amino
terminal 36 amino acids of FANCA form a novel interaction with the
FANCG protein and identified two different carboxy terminal regions of
FANCG (encompassing amino acids 400-475 and 585-622) required for FANCA
binding. However, these investigators failed to detect FANCC binding in
the FANCA/FANCG complex, although the FANCC interaction has been
verified by other independent investigators.25,26 Our
results confirm the contribution of FANCG amino acids 400-428 to FANCA
binding and demonstrate that the carboxy terminus of FANCG (amino acid
585-622) is required for FANCC binding and optimum FANCA binding in the
complex. Indeed, our data demonstrate that FANCC binding stabilizes the
FANCA/FANCG interaction, because the FANCG1749
We thank members of the D'Andrea laboratory for helpful discussions.
Submitted February 3, 2000; accepted April 27, 2000.
Supported by grants RO1HL52725-04 and PO1HL54785-04 from the National Institutes of Health. A.D.D. is a Scholar of the Leukemia Society of America. I.G.-H. is supported by the Cancer Research Institute.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Alan D. D'Andrea, Dana-Farber Cancer Institute, Division of Pediatric Oncology, Harvard Medical School, 44 Binney St, Boston, MA 02115; e-mail: alan_dandrea{at}dfci.harvard.edu.
1. Auerbach AD, Buchwald M, Joenje H. Fanconi anemia. In: Vogelstein B,Kinzler KW, eds. Genetics of Human Cancer. New York: McGraw Hill; 1997. 2. Garcia-Higuera I, Kuang Y, D'Andrea AD. The molecular and cellular biology of Fanconi anemia. Curr Opin Hematol. 1999;2:83-88. 3. Joenje H, Oostra AB, Wijker M, et al. Evidence for at least eight Fanconi anemia genes. Am J Hum Genet. 1997;61:940-944[Medline] [Order article via Infotrieve]. 4. de Winter JP, Waisfisz Q, Rooimans MA, et al. The Fanconi anaemia group G gene is identical with human XRCC9. Nat Genet. 1998;20:281-283[Medline] [Order article via Infotrieve]. 5. Strathdee CA, Gavish H, Shannon WR, Buchwald M. Cloning of cDNAs for Fanconi's anaemia by functional complementation. Nature 1992;356:763-767[Medline] [Order article via Infotrieve]. 6. The Fanconi Anemia Consortium. Positional cloning of the Fanconi anaemia group A gene. Nat Genet. 1996;14:324-328[Medline] [Order article via Infotrieve]. 7. Lo Ten Foe JR, Rooimans MA, Bosnoyan-Collins L, et al. Expression cloning of a cDNA for the major Fanconi anemia gene, FAA. Nat Genet. 1996;14:320-323[Medline] [Order article via Infotrieve]. 8. de Winter JP, Rooimans MA, van der Weel L, et al. The Fanconi anemia complementation gene FANCF encodes a novel protein with homology to ROM. Nat Genet. 2000;24:15-16[Medline] [Order article via Infotrieve].
9.
Liu N, Lamerdin JE, Tucker JD, et al.
The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells.
Proc Natl Acad Sci U S A.
1997;94:9232-9237 10. Whitney M, Thayer M, Reifsteck C, et al. Microcell mediated chromosome transfer maps the Fanconi anemia group D gene to chromosome 3p. Nat Genet. 1995;11:341-343[Medline] [Order article via Infotrieve]. 11. Waisfisz Q, Saar K, Morgan NV, et al. The Fanconi anemia group E gene, FANCE, maps to chromosome 6p. Am J Hum Genet. 1999;64:1400-1405[Medline] [Order article via Infotrieve].
12.
Liu J, Buchwald M, Walsh CE, Young NS.
Fanconi anemia and novel strategies for therapy.
Blood.
1994;84:3995-4007
13.
D'Andrea AD, Grompe M.
Molecular biology of Fanconi anemia: implications for diagnosis and therapy.
Blood.
1997;90:1725-1736 14. Korkina LG, Samochatova EV, Maschan AA, Suslova TB, Cheremisina ZP, Afanas'ev IB. Release of active oxygen radicals by leukocytes of Fanconi anemia patients. J Leukoc Biol. 1992;52:357-362[Abstract]. 15. Schlindler D, Hoehn H. Fanconi anemia mutation causes cellular susceptibility to ambient oxygen. Am J Hum Genet. 1988;43:429-435[Medline] [Order article via Infotrieve]. 16. Hojo ET, van Dieman PC, Darroudi F, Natarajan AT. Spontaneous chromosomal aberrations in Fanconi anaemia, ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique [published erratum appears in Mutat Res. 1995;334:268-270]. Mutat Res. 1995;334:59-69[Medline] [Order article via Infotrieve]. 17. Kaiser TN, Lojewski A, Dougherty C, Juergens L, Sahar E, Latt SA. Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment. Cytometry. 1982;2:291-297[Medline] [Order article via Infotrieve]. 18. Kubbies M, Schindler D, Hoehn H, Schinzel A, Rabinovich PS. Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells. Am J Hum Genet. 1985;37:1022-1030[Medline] [Order article via Infotrieve].
19.
Whitney MA, Royle G, Low MJ, et al.
Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene.
Blood.
1996;88:49-58
20.
Rathbun R, Faulkner G, Ostroski M, et al.
Inactivation of the Fanconi anemia group C gene augments interferon-gamma induced apoptotic responses in hematopoietic cells.
Blood.
1997;90:974 21. Smith J, Andrau JC, Kallenbach S, Laquerbe A, Doyen N, Papadopoulo D. Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia. J Mol Biol. 1998;281:815-825[Medline] [Order article via Infotrieve]. 22. Escarceller M, Rousset S, Moustacchi E, Papadopoulo D. The fidelity of double strand breaks processing is impaired in complementation groups B and D of Fanconi anemia, a genetic instability syndrome. Somat Cell Mol Genet. 1997;23:401-411[Medline] [Order article via Infotrieve]. 23. D'Andrea AD. Fanconi anaemia forges a novel pathway. Nat Genet. 1996;14:240-242[Medline] [Order article via Infotrieve].
24.
Garcia-Higuera I, Kuang Y, Naf D, Wasik J, D'Andrea AD.
Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex.
Mol Cell Biol.
1999;19:4866-4873
25.
McMahon LW, Walsh CE, Lambert MW.
Human
26.
Christianson TA, Bagby GC.
FANCA protein binds FANCC and FANCG proteins in an intracellular complex.
Blood.
2000;95:725-726
27.
Yamashita T, Kupfer GM, Naf D, et al.
The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation.
Proc Natl Acad Sci U S A.
1998;95:13085-13090 28. Kupfer GM, Naf D, Suliman A, Pulsipher M, D'Andrea AD. The Fanconi anemia proteins, FAA and FAC, interact to form a nuclear complex. Nat Genet. 1997;17:487-490[Medline] [Order article via Infotrieve]. 29. Kupfer G, Naf D, Garcia-Higuera I, et al. A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation. Exp Hematol. 1999;27:587-593[Medline] [Order article via Infotrieve].
30.
Naf D, Kupfer GM, Suliman A, Lambert K, D'Andrea AD.
Functional activity of the Fanconi anemia protein, FAA, requires FAC binding and nuclear localization.
Mol Cell Biol.
1998;18:5952-5960
31.
Ory D, Neugeboren B, Mulligan R.
A stable human-derived packaging cell line for production of high-titer retrovirus/vesicular stomatitis virus G pseudotypes.
Proc Natl Acad Sci U S A.
1996;93:11400-11406
32.
Kuang Y, Wu Y, Jiang H, Wu D.
Selective G protein coupling by chemokine receptors.
J Biol Chem.
1996;271:3975-3978 33. Pulsipher M, Kupfer GM, Naf D, et al. Subtyping analysis of Fanconi anemia by immunoblotting and retroviral gene transfer. Mol Med. 1998;4:468-479[Medline] [Order article via Infotrieve].
34.
Lightfoot J, Alon N, Bosnoyan-Collins L, Buchwald M.
Characterization of regions functional in the nuclear localization of the Fanconi anemia group A protein.
Hum Mol Genet.
1999;8:1007-1015
35.
Waisfisz Q, De Wintr JP, Kruyt FA, et al.
A physical complex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA.
Proc Natl Acad Sci U S A.
1999;96:10320-10325
36.
Durfee T, Becherer K, Chen PL, et al.
The retinoblastoma protein associates with the protein phosphatase type I catalytic subunit.
Genes Dev.
1993;7:555-569
37.
Keegan KS, Holtzman DA, Plug AW, et al.
The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes.
Genes Dev.
1996;10:2423-2437 38. Ma J, Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987;48:847-853[Medline] [Order article via Infotrieve].
39.
Chen CF, Li S, Chen Y, Phang-Lang C, Sharp D, Wen-Hwa L.
The nuclear localization sequences of the BRCA1 protein interact with the importin-
40.
Reuter T, Herterich S, Bernhard O, Hoehn H, Gross HJ.
Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system.
Blood.
2000;95:719-720 41. Ferrigno P, Silver PA. Analysis of nuclear transport in vivo. Methods Cell Biol. 1999;58:107-122[Medline] [Order article via Infotrieve].
42.
Gorlich D, Dabrowski M, Bischoff FR, et al.
A novel class of RanGTP binding proteins.
J Cell Biol.
1997;138:65-80 43. Mathew CG, Morgan N, Tipping A, Huber P. Molecular pathology and functional analysis in Fanconi anemia. Am J Hum Genet. 1998;63:A373. 44. Waisfisz Q, de Winter JP, Kruyt F, et al. A physical complex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA. Proc Natl Acad Sci U S A. 1999;96:10320-10325.
45.
Kruyt FA, Abou-Zahr F, Mok H, Youssoufian H.
Resistance to mitomycin C requires direct interaction between the Fanconi anemia protein FANCA and FANCG in the nucleus through an arginine-rich domain.
J Biol Chem.
1999;274:34212-34218
© 2000 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  J. M. Kim, Y. Kee, A. Gurtan, and A. D. D'Andrea Cell cycle-dependent chromatin loading of the Fanconi anemia core complex by FANCM/FAAP24 Blood, May 15, 2008; 111(10): 5215 - 5222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Pejovic, J. E. Yates, H. Y. Liu, L. E. Hays, Y. Akkari, Y. Torimaru, W. Keeble, R. K. Rathbun, W. H. Rodgers, A. E. Bale, et al. Cytogenetic instability in ovarian epithelial cells from women at risk of ovarian cancer. Cancer Res., September 15, 2006; 66(18): 9017 - 9025. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. van der Heijden, J. R. Brody, D. A. Dezentje, E. Gallmeier, S. C. Cunningham, M. J. Swartz, A. M. DeMarzo, G. J. A. Offerhaus, W. H. Isacoff, R. H. Hruban, et al. In vivo Therapeutic Responses Contingent on Fanconi Anemia/BRCA2 Status of the Tumor Clin. Cancer Res., October 15, 2005; 11(20): 7508 - 7515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Leveille, E. Blom, A. L. Medhurst, P. Bier, E. H. Laghmani, M. Johnson, M. A. Rooimans, A. Sobeck, Q. Waisfisz, F. Arwert, et al. The Fanconi Anemia Gene Product FANCF Is a Flexible Adaptor Protein J. Biol. Chem., September 17, 2004; 279(38): 39421 - 39430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Van der Heijden, J. R. Brody, E. Gallmeier, S. C. Cunningham, D. A. Dezentje, D. Shen, R. H. Hruban, and S. E. Kern Functional Defects in the Fanconi Anemia Pathway in Pancreatic Cancer Cells Am. J. Pathol., August 1, 2004; 165(2): 651 - 657. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Lensch, M. Tischkowitz, T. A. Christianson, C. A. Reifsteck, S. A. Speckhart, P. M. Jakobs, M. E. O'Dwyer, S. B. Olson, M. M. Le Beau, S. V. Hodgson, et al. Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia Blood, July 1, 2003; 102(1): 7 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Gordon and M. Buchwald Fanconi anemia protein complex: mapping protein interactions in the yeast 2- and 3-hybrid systems Blood, July 1, 2003; 102(1): 136 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Digweed, S. Rothe, I. Demuth, R. Scholz, D. Schindler, M. Stumm, M. Grompe, A. Jordan, and K. Sperling Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia Carcinogenesis, July 1, 2002; 23(7): 1121 - 1126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Wilson, M. A. Johnson, A. P. Stuckert, K. L. Trueman, S. May, P. E. Bryant, R. E. Meyn, A. D. D'Andrea, and N. J. Jones 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, December 1, 2001; 22(12): 1939 - 1946. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
and interferon
.
). NLS, nuclear localization signal; Lz,
leucine zipper. (B) COS cells were cotransfected with the indicated
FANCA cDNA mutants and the full-length FANCG cDNA. Immunoprecipitation
(lanes 1-6) was performed with an anti-FANCG antiserum (upper blot) and
an anti-HA antibody (middle blot). Immunoblotting was done with an
anti-HA monoclonal antibody or with an anti-FANCG antiserum, as
indicated. WCE denotes whole cell extract.
), EUFA316 cells
transduced with FANCG(wt) (
), with FANCG 1-428 (
), with
FANCG
), or with FANCG1749
). Similar results were
obtained when the FANCG cDNAs were transfected into the FA-G fibroblast
line, FAG326SV (data not shown).
, or nucleoporins,
remains to be determined.