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RED CELLS
From the Department of Pediatric Oncology, Dana-Farber
Cancer Institute, Harvard Medical School, Boston, MA.
Fanconi anemia (FA) is an autosomal recessive cancer
susceptibility syndrome with 8 complementation groups. Four of the FA genes have been cloned, and at least 3 of the encoded proteins, FANCA,
FANCC, and FANCG/XRCC9, interact in a multisubunit protein complex. The
FANCG protein binds directly to the amino terminal nuclear localization
sequence (NLS) of FANCA, suggesting that FANCG plays a role in
regulating FANCA nuclear accumulation. In the current study the
functional consequences of FANCG/FANCA binding were examined.
Correction of an FA-G cell line with the FANCG complementary DNA (cDNA)
resulted in FANCA/FANCG binding, prolongation of the cellular half-life
of FANCA, and an increase in the nuclear accumulation of the FA protein
complex. Similar results were obtained upon correction of an FA-A cell
line, with a reciprocal increase in the half-life of FANCG.
Patient-derived mutant forms of FANCA, containing an intact NLS
sequence but point mutations in the carboxy-terminal leucine zipper
region, bound FANCG in the cytoplasm. The mutant forms failed to
translocate to the nucleus of transduced cells, thereby suggesting a
model of coordinated binding and nuclear translocation. These results
demonstrate that the FANCA/FANCG interaction is required to maintain
the cellular levels of both proteins. Moreover, at least one function
of FANCG and FANCA is to regulate the nuclear accumulation of the FA
protein complex. Failure to accumulate the nuclear FA protein complex
results in the characteristic spectrum of clinical and cellular
abnormalities observed in FA.
(Blood. 2000;96:3224-3230) 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.1,2 Based on somatic cell fusion
studies, FA comprises 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 was found to be
identical to human XRCC9, a gene proposed to be involved in
cell cycle regulation or post-replication repair.9 Two
additional genes, 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. However, based on the similar clinical and cellular phenotypes observed
among the 8 FA complementation groups, the FA genes appear to
participate in a common cellular pathway.
Cells from FA patients display a broad range of
abnormalities12,13 including hypersensitivity to DNA
cross-linking agents and oxygen radicals,14,15 spontaneous
chromosome breakage,16 cell cycle abnormalities manifested
by a prolongation in the G2 phase of the cell cycle,17,18
and increased cellular sensitivity to the apoptotic effects of tumor
necrosis factor (TNF)- Increasing evidence demonstrates that the FA proteins cooperate in a
novel cellular pathway.23 FANCA, FANCG, and FANCC bind and
interact in a protein complex, which is observed in the cytoplasm and
the nucleus of normal cells.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 gene,8 may regulate the
formation of the complex, perhaps by serving as adaptor proteins or
enzymes regulating protein complex assembly. The FA-D complementation
group is distinct from other FA groups.27 In FA-D cells
the FA protein complex assembles normally, which suggests that the
FANCD protein product may function downstream or independently of the complex.
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-terminal of FANCC, such as the patient-derived FANCC-L554P
mutation,5 block complex formation.28
Similarly, point mutations in the carboxy-terminal leucine zipper
region of FANCA, such as the histidine (His) 1110 to proline (Pro)
mutation,29 give rise to a FANCA protein that is defective
in FANCC binding, phosphorylation, and nuclear localization.
Point mutations thereby help to define many of the normal
biochemical events in the FA pathway.27
Recent studies suggest that the FANCA and FANCG proteins participate in
a direct interaction.24,30,31 The amino terminal NLS of
FANCA, containing amino acids 1-36, forms a direct binding interaction
with FANCG.31,32 In contrast, the interaction of FANCC
with the FA protein complex appears to be indirect and requires additional post-translational modifications or adaptor
proteins.27
In the current study we examined the functional consequences of
FANCG/FANCA binding. Interestingly, we found that the FANCG and FANCA
proteins stabilize each other and promote the nuclear accumulation of
the FA protein complex. Moreover, the binding of FANCA and FANCG
initially occurs in the cytoplasm of normal cells prior to the
regulated transport of the complex to the nucleus. Accumulation of this
nuclear complex is required for the maintenance of chromosome stability.
Cell culture
Retroviral infection of FA cell lines
FA lymphoblasts were infected with the various pMMP supernatants for 4 hours in the presence of 8 µg/mL polybrene (Sigma Chemical Co, St Louis, MO). Infected cells were washed free of viral supernatant and resuspended in growth media. After 48 hours the cells were transferred to media containing 1 µg/mL puromycin. The dead cells were removed over a Ficoll cushion after 5 days, and the surviving cells were grown under continuous selection in puromycin. Analysis of FANCA levels by flow cytometry The indicated FA lymphoblasts were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at room temperature and permeabilized for 10 minutes using 0.3% Triton X-100 in PBS. After washing, the cells were resuspended in blocking buffer (3% bovine serum albumin [BSA] and 0.1% NP-40 in PBS) and incubated for 1 hour prior to the addition of anti-FANCA or anti-FANCG affinity-purified rabbit antisera at a 1:100 dilution. After either 2 hours at room temperature or 15 hours at 4°C, the cells were washed 3 times with PBS and incubated for an additional hour with a fluorescein isothiocyanate (FITC)-conjugated goat antirabbit antibody (Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1:400 in blocking buffer. The cells were rinsed again with PBS and resuspended in PBS with 2% BSA, and cellular fluorescence was measured using a fluorescence-activated cell sorter (FACScan; Becton Dickinson, San Jose, CA). Data analysis was subsequently performed with the software program CellQuest (Becton Dickinson).Immunoprecipitation and pulse chase experiments Whole cell extracts were prepared in lysis buffer comprising 50 mmol/L Tris HCl (tris[hydroxymethyl] aminomethane-hydrochloride) (pH 7.4), 150 mmol/L sodium chloride (NaCl), and 1% (vol/vol) Triton X-100. The buffer was supplemented with protease inhibitors (1 µg/mL leupeptin and pepstatin, 2 µg/mL aprotinin, and 1 mmol/L phenylmethylsulfonylfluoride) and phosphatase inhibitors (1 mmol/L sodium orthovanadate and 10 mmol/L sodium fluoride). Immunoprecipitation and Western blotting were performed as previously described24 using affinity-purified rabbit antisera raised against the C-termini of FANCA, FANCC, or FANCG.24,28 For pulse-chase experiments the cells were metabolically labeled with sulfur 35 (35S)-methionine for 30 minutes, washed, and chased in cold (unlabeled) medium for the indicated time periods. The cells were then lysed in lysis buffer, and the labeled proteins were immunoprecipitated with anti-FANCA or anti-FANCG affinity-purified antisera and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography.Cell fractionation Both lymphoblasts and fibroblasts were fractionated into cytoplasmic and nuclear proteins by hypotonic swelling. This was followed by Dounce homogenization, essentially as previously described.36Generation of human FANCA complementary DNA mutant constructs Wild type and mutant FANCA complementary DNA (cDNA) was subcloned into the retroviral expression vector pMMP34 as previously described.33 Mutations in the FANCA cDNA were introduced by polymerase chain reaction (PCR) with Pfu polymerase (Stratagene, La Jolla, CA), and cDNA inserts were verified by DNA sequencing.Immunofluorescence microscopy Immunofluorescence microscopy of human fibroblasts was performed as previously described.33
FANCA/FANCG binding regulates the stability of the FA protein
complex. We initially demonstrated that FANCG increases FANCA protein
levels by using a flow cytometric approach (Figure
1) which allows analysis of the cellular
contents of a given protein without the need for cell lysis. FA-A
lymphoblasts (HSC72) failed to express the FANCA protein28
and showed little staining with an anti-FANCA antisera (Figure 1A,
upper panel). Functional complementation of these cells with the FANCA
cDNA resulted in the correction of MMC sensitivity24 and an
increase in the anti-FANCA fluorescence signal (Figure 1A). The FA-G
lymphoblast EUFA 316, which failed to express the FANCG protein, also
showed little anti-FANCA staining (Figure 1B). However, correction of
the MMC sensitivity of these cells by transduction of the FANCG cDNA
resulted in a significant increase in the FANCA protein levels (Figure
1B). Moreover, functional complementation of the FA-A cells (HSC72)
also lead to a significant increase in the anti-FANCG fluorescence
signal (Figure 1C), which suggests that the positive effect on the
expression level was reciprocal between the 2 FA proteins.
These results were also confirmed by immunoblot analysis (Figure
2). Again, correction of FA-A cells with
FANCA resulted in a net increase in the cellular FANCG levels (Figure
2, lanes 2 and 3); correction of FA-G cells with FANCG resulted in a
net increase in the cellular FANCA levels (Figure 2, lanes 4 and 5). We
next sought to determine whether the induction in the FANCA and FANCG
protein level was either a consequence of the phenotypic correction of
the cells or directly related to the FANCA/FANCG interaction. For this
purpose, in the FA-A lymphoblasts (HSC72) we expressed 2 previously characterized nonfunctional mutant forms of
FANCA24 (Figure 2, lanes 6-9): one that retains the ability to interact with FANCG (FANCA-H1110P) and an amino-terminal
truncation mutant lacking the FANCG binding region (FANCA-
Regulation of the expression level of the FA protein complex could
result from one or more mechanisms including transcriptional up-regulation, translational up-regulation, or decreased turnover of a
messenger RNA (mRNA) or protein. To directly test the effect of FANCG
on FANCA protein turnover, we performed pulse-chase experiments (Figure
3). The patient-derived FA-G cell line,
EUFA316, was sensitive to mitomycin C and failed to express the FANCG
protein.24,37 In these cells the newly synthesized FANCA
protein was unstable, with a protein half-life of 1 hour (Figure 3A).
In contrast, in EUFA316 cells stably transfected and functionally
corrected with FANCG, the half-life of FANCA was more than 10 hours,
which suggests that the FANCG protein directly stabilizes the FANCA
protein. By a similar analysis, the FANCA protein was shown to
complement FA-A cells and to promote the stability of the FANCG
protein, as evidenced by an 8- to 10-fold increase in its half-life
(Figure 3B). Once again, to discriminate between functional
complementation and a more direct effect of the 2 FA proteins on each
other, we examined the stability of FANCA and FANCG in a
patient-derived MMC-sensitive FA-C cell line (PD4). These cells have no
endogenous FANCC protein but have normal FANCA/FANCG binding (Figure
3C).24 Consistent with our previous observations, the
half-lives of both proteins in these MMC-sensitive cells were
comparable to those obtained in the corrected FA-A and FA-G cells.
Taken together, our results suggest that the FANCA/FANCG interaction
determines the stability of the 2 FA proteins and is thus essential to
maintain the cellular levels of the FA protein complex.
FANCG binding promotes the nuclear accumulation of the FA protein
complex. Recent studies have demonstrated that FANCG binds directly to
FANCA in the region of the FANCA NLS,31,32 which suggests
that FANCG could play a role in modulating the subcellular localization
of FANCA. We therefore tested the effect of FANCA/FANCG binding on the
nuclear accumulation of FANCA and FANCG (Figure 4). Based on cell fractionation studies,
FA-A lymphoblasts (HSC72) expressed little FANCG in the nucleus (Figure
4, lane 4) compared to normal lymphoblasts (Figure 4, lane 2).
Correction of the FA-A lymphoblasts with FANCA resulted in an increase
in the total cellular level of FANCG and, more importantly, in the
ratio of nuclear to cytoplasmic FANCG protein (Figure 4, compare lanes
3 and 4 to lanes 5 and 6). Similarly, correction of FA-G cells with
FANCG lead to an increase in nuclear FANCA levels (Figure 4, compare lanes 12 and 14). Interestingly, in corrected FA-A (Figure 4, lanes 5 and 6) and FA-G cells (Figure 4, lanes 13 and 14), the ratio of nuclear
to cytoplasmic FANCC was also higher than in the corresponding mutant
cells (Figure 4, lanes 3 and 4 and lanes 11 and 12). This ratio
suggests that the FANCA/FANCG interaction may be important for nuclear
accumulation of the whole FA protein complex. FA lymphoblasts from
other FA complementation groups, such as FA-C (PD-4 cells), also had
decreased levels of FANCA and FANCG protein in the nucleus (Figure 4,
lane 8) that were restored after complementation with the FANCC cDNA
(Figure 4, lane 10). These results demonstrate that formation of the
FANCA/FANCG oligomer is necessary but not sufficient for the proper
accumulation of the FA proteins in the nucleus. Other FA protein
products, such as FANCC, are also required for nuclear accumulation of
the FA protein complex. Because we have previously established that the
FANCA/FANCG interaction is a prerequisite for FANCC binding, we
hypothesize that it is the formation of the multisubunit complex containing FANCA, FANCG, and FANCC which determines the presence of the
FA proteins in the nuclear compartment. Whether the effect is on
nuclear import/export rates or on protein stability remains to be
determined.
Patient-derived mutant forms of the FANCA protein bind and retain
FANCG in the cytoplasm. Previous studies have identified patient-derived missense mutations in the FANCA gene that
result in the expression of mutant FANCA proteins.29,38
The FANCA-H1110P and FANCA-R1117G mutant proteins, for example, bind
FANCG but fail to correct the MMC sensitivity of FA-A cells. To further explore the consequences of the FANCA/FANCG interaction, we analyzed the cellular localization of the FANCA/FANCG protein complex in FA-A
fibroblasts transduced with the cDNA encoding these mutant FANCA
proteins (Figure 5). The wild type FANCA
protein and wild type FANCG protein colocalized to the nucleus, as
previously described (Figure 5, column 3). In contrast, the
FANCA-H1110P and FANCA-R1117G mutant proteins were localized primarily
to the cytoplasm of transduced GM6914 cells (Figure 5, columns 4 and
5). Interestingly, the expression of these mutant FANCA proteins
resulted in the retention of the wild type FANCG protein in the
cytoplasm.
We previously described another mutant form of the FANCA protein called FANCA-NLS-Mut2.33 In this protein all of the basic amino acids of the bipartite NLS sequence were mutated. In our previous study, FANCA-NLS Mut2 failed to complement FA-A cells,33 to translocate to the nucleus of transduced FA-A cells,33 and to bind to FANCG.24 In our new study FANCA-NLS-Mut2 again localized to the cytoplasm (Figure 5, column 6), but FANCG was observed in the nucleus. These results demonstrate that when FANCG is overexpressed, it can translocate to the nucleus independently of FANCA. However, in a normal cell with endogenous levels of FANCA and FANCG, this may not be the preferred pathway. Moreover, given the short half-life of FANCG in the absence of FANCA binding (Figure 3), endogenous FANCG may translocate but fail to accumulate in the nucleus. To confirm these results with an endogenous FANCG protein, we performed
a series of cellular fractionation experiments (Figure 6). In FA-A fibroblasts we observed
little or no FANCA or FANCG protein in the cytoplasm and nucleus
(Figure 6, lanes 3 and 4). Complementation of these cells with FANCA
resulted in the expression of FANCA and FANCG in the cytoplasm and the
nucleus (Figure 6, lanes 5 and 6). In contrast, forced expression of
FANCA-H1110P resulted in the expression of little or no FANCG in the
nucleus, which is consistent with the absence of nuclear FANCA or FANCG immunofluorescence observed in these cells (Figure 5). Similar results
were obtained after expression of the FANCA-R1117G (data not shown).
Several lines of evidence demonstrate that the FA proteins, including FANCA, FANCG, and FANCC, interact in a common cellular pathway and lead 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 are required for the assembly and stability of the nuclear FA complex,27 suggesting that many FA genes are involved in the same cellular pathway. While the presence of the FANCC protein product in the nuclear FA complex has been controversial,31 the nuclear complex has recently been confirmed by other investigators.25,26 In the current study we demonstrated that the functional consequence of the FANCG/FANCA interaction is stabilization and increased nuclear accumulation of the FA protein complex. Formation of the FANCA/FANCG complex results in the stabilization of both proteins. Several mechanisms could account for this effect. First, FANCA/FANCG binding may protect the 2 proteins from proteolysis by masking the proteolytic cleavage sites of the proteins. At present the intracellular proteases responsible for FANCA or FANCG degradation remain unknown. However, we attempted to address this question by using the specific proteosome inhibitor lactacystin. Following drug treatment a significant increase in the level of the p53 protein was observed, but there were no changes detected in the FANCA or FANCG proteins (data not shown). This suggests that the proteosome is not involved in the rapid turnover of these proteins. Second, the FANCA/FANCG interaction may promote translocation of the complex to an intracellular site, such as a nuclear subcompartment, where the 2 proteins are more stable. Third, FANCA/FANCG oligomerization may promote the binding of other FA protein subunits, such as FANCC or FANCF, which further stabilize the multisubunit complex. One or more of these 3 cellular mechanisms may serve to increase the stability of FANCA and FANCG. The systematic analysis of the half-life of the FANCA/FANCG complex in cell lines from other FA complementation groups may help distinguish among these possible mechanisms. In contrast, the cellular level of the FANCC protein is not strongly dependent on the level of FANCA and FANCG in the cell. Instead, FANCC levels may be regulated by GRP94 chaperone binding39 and/or by post-translational modifications.40 Our results demonstrate that the FANCG expression promotes the
accumulation of FANCA in the nucleus. Mutant forms of FANCG, which fail
to bind FANCA, also fail to promote nuclear accumulation of FANCA (data
not shown). Because FANCG has been shown to bind specifically to the
NLS region of FANCA,41 it is reasonable to assume that
FANCG may play an active role in modulating the nuclear import and/or
export processes of the FA protein complex. For instance, FANCG may
increase nuclear uptake by acting as an importin protein that
recognizes the NLS region of FANCA and allows the interaction with
nuclear pore complexes (NPCs)42 or by cooperating with
importin- In transduced fibroblasts, FANCA and FANCG proteins are capable of translocating to the nucleus independently: FANCG translocates to the nucleus of FA-A cells in the absence of functional FANCA protein, and FANCA translocates to the nucleus of FA-G cells in the absence of functional FANCG protein (I.G.-H., unpublished observation, April 2000). Whether this independent nuclear translocation occurs in normal cells and to what extent it occurs, if at all, remain to be determined. However, because FANCA and FANCG depend on each other for their stability (Figure 3), it is unlikely that at normal physiological levels, neither of the 2 proteins accumulate in the nuclear compartment. Our fractionation data would agree with this idea. Moreover, preliminary evidence suggests that the half-life of nuclear FANCA is comparable to that of the cytoplasmic form (unpublished observation). Therefore, FANCA/FANCG binding appears to be a more important determinant of FA complex stability than does subcellular localization. Our data support a model of the FA pathway in which FANCG binds FANCA
in the cytoplasm, before their coordinated translocation to the nucleus
(Figure 7). In support of this model,
some FA patient-derived mutant forms of the FANCA protein (FANCA-H111OP
and FANCA-R1117G) bind FANCG in the cytoplasm but are arrested in the
pathway, before their translocation and accumulation in the nucleus.
Interestingly, these point mutations also ablate the phosphorylation of
the FANCA protein, the binding of the FANCC protein, and the
accumulation of the stable nuclear complex.29 How the
FANCA point mutations H111OP and R1117G block the downstream events in
the FA pathway remains unknown, but several mechanisms can be
considered. For instance, these point mutations, which are only 7 amino
acids apart in the leucine zipper region of FANCA, may block the
phosphorylation of FANCA by preventing binding of the relevant FANCA
protein kinases (FANCA-PKs). Alternatively, these mutations may prevent
the binding of additional protein subunits, perhaps encoded by other FA
genes. Regardless of the correct hypothesis, these data
demonstrate that several structural features of FANCA, including a
functional amino-terminal NLS and an intact carboxy-terminal leucine
zipper region, are required for normal nuclear translocation.
This model generates new testable hypotheses. For instance, it will be important to determine whether the assembly and transport of the FANCA/FANCG complex is constitutive or regulated by external environmental cues or cell cycle cues. For instance, bifunctional alkylating agents may affect the complex assembly or accumulation. Also, identification of the cytoplasmic kinase of FANCA (FANCA-PK) may elucidate a critical level of regulation of the FA protein complex assembly and function. Finally, while the accumulation of the FA protein complex appears to be a critical step in the FA pathway, the precise biochemical function of this complex in the nucleus remains unknown. Increasing evidence suggests that FA cells are defective in DNA repair, and the FA complex may play a direct or indirect role in the repair of DNA damage. Also, the FA protein complex assembles normally in FA-D cells, suggesting that the product of the FANCD gene functions downstream or independently of the FA protein complex assembly. Whether the FA protein complex functions by modulating the structure and function of the recently cloned FANCD protein product (Timmers et al, unpublished data) remains unknown.
We thank members of the D'Andrea laboratory (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) for helpful discussions.
Supported by grants RO1HL52725-04 and PO1HL54785-04 from the National Institutes of Health, Bethesda, MD, and a post-doctoral grant (G.-H.) from the Cancer Research Institute. A.D.D. is a Scholar of the Leukemia Society of America.
Submitted April 12, 2000; accepted June 20, 2000.
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 Street, Boston, MA 02115; e-mail: alan_dandrea{at}dfci.harvard.edu.
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  F. Yuan, J. El Hokayem, W. Zhou, and Y. Zhang FANCI Protein Binds to DNA and Interacts with FANCD2 to Recognize Branched Structures J. Biol. Chem., September 4, 2009; 284(36): 24443 - 24452. [Abstract] [Full Text] [PDF]
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C. S. Tremblay, C. C. Huard, F.-F. Huang, O. Habi, V. Bourdages, G. Levesque, and M. Carreau The Fanconi Anemia Core Complex Acts as a Transcriptional Co-regulator in Hairy Enhancer of Split 1 Signaling J. Biol. Chem., May 15, 2009; 284(20): 13384 - 13395. [Abstract] [Full Text] [PDF]
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 Y. Kee, J. M. Kim, and A. D'Andrea Regulated degradation of FANCM in the Fanconi anemia pathway during mitosis Genes & Dev., March 1, 2009; 23(5): 555 - 560. [Abstract] [Full Text] [PDF]
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 C. S. Tremblay, F. F. Huang, O. Habi, C. C. Huard, C. Godin, G. Levesque, and M. Carreau HES1 is a novel interactor of the Fanconi anemia core complex Blood, September 1, 2008; 112(5): 2062 - 2070. [Abstract] [Full Text] [PDF]
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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]
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 S. Stone, A. Sobeck, M. van Kogelenberg, B. de Graaf, H. Joenje, J. Christian, and M. E. Hoatlin Identification, developmental expression and regulation of the Xenopus ortholog of human FANCG/XRCC9 Genes Cells, July 1, 2007; 12(7): 841 - 851. [Abstract] [Full Text] [PDF]
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T. Oda, T. Hayano, H. Miyaso, N. Takahashi, and T. Yamashita Hsp90 regulates the Fanconi anemia DNA damage response pathway Blood, June 1, 2007; 109(11): 5016 - 5026. [Abstract] [Full Text] [PDF]
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A. M. Gurtan, P. Stuckert, and A. D. D'Andrea The WD40 Repeats of FANCL Are Required for Fanconi Anemia Core Complex Assembly J. Biol. Chem., April 21, 2006; 281(16): 10896 - 10905. [Abstract] [Full Text] [PDF]
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 A. Sobeck, S. Stone, V. Costanzo, B. de Graaf, T. Reuter, J. de Winter, M. Wallisch, Y. Akkari, S. Olson, W. Wang, et al. Fanconi Anemia Proteins Are Required To Prevent Accumulation of Replication-Associated DNA Double-Strand Breaks Mol. Cell. Biol., January 15, 2006; 26(2): 425 - 437. [Abstract] [Full Text] [PDF]
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R. D. Kennedy and A. D. D'Andrea The Fanconi Anemia/BRCA pathway: new faces in the crowd Genes & Dev., December 15, 2005; 19(24): 2925 - 2940. [Abstract] [Full Text] [PDF]
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S. M. Gordon, N. Alon, and M. Buchwald FANCC, FANCE, and FANCD2 Form a Ternary Complex Essential to the Integrity of the Fanconi Anemia DNA Damage Response Pathway J. Biol. Chem., October 28, 2005; 280(43): 36118 - 36125. [Abstract] [Full Text] [PDF]
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 M. Ferrer, J. A. Rodriguez, E. A. Spierings, J. P. de Winter, G. Giaccone, and F. A.E. Kruyt Identification of multiple nuclear export sequences in Fanconi anemia group A protein that contribute to CRM1-dependent nuclear export Hum. Mol. Genet., May 15, 2005; 14(10): 1271 - 1281. [Abstract] [Full Text] [PDF]
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F. Qiao, J. Mi, J. B. Wilson, G. Zhi, N. R. Bucheimer, N. J. Jones, and G. M. Kupfer Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway J. Biol. Chem., October 29, 2004; 279(44): 46035 - 46045. [Abstract] [Full Text] [PDF]
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J. Mi, F. Qiao, J. B. Wilson, A. A. High, M. J. Schroeder, P. T. Stukenberg, A. Moss, J. Shabanowitz, D. F. Hunt, N. J. Jones, et al. FANCG Is Phosphorylated at Serines 383 and 387 during Mitosis Mol. Cell. Biol., October 1, 2004; 24(19): 8576 - 8585. [Abstract] [Full Text] [PDF]
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S.-J. Park, S. L. M. Ciccone, B. D. Beck, B. Hwang, B. Freie, D. W. Clapp, and S.-H. Lee Oxidative Stress/Damage Induces Multimerization and Interaction of Fanconi Anemia Proteins J. Biol. Chem., July 16, 2004; 279(29): 30053 - 30059. [Abstract] [Full Text] [PDF]
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 J. E. Lamerdin, N. A. Yamada, J. W. George, B. Souza, A. T. Christian, N. J. Jones, and L. H. Thompson Characterization of the hamster FancG/Xrcc9 gene and mutations in CHO UV40 and NM3 Mutagenesis, May 1, 2004; 19(3): 237 - 244. [Abstract] [Full Text] [PDF]
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I. Brodeur, I. Goulet, C. S. Tremblay, C. Charbonneau, M.-C. Delisle, C. Godin, C. Huard, E. W. Khandjian, M. Buchwald, G. Levesque, et al. Regulation of the Fanconi Anemia Group C Protein through Proteolytic Modification J. Biol. Chem., February 6, 2004; 279(6): 4713 - 4720. [Abstract] [Full Text] [PDF]
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K. Yamamoto, M. Ishiai, N. Matsushita, H. Arakawa, J. E. Lamerdin, J.-M. Buerstedde, M. Tanimoto, M. Harada, L. H. Thompson, and M. Takata Fanconi Anemia FANCG Protein in Mitigating Radiation- and Enzyme-Induced DNA Double-Strand Breaks by Homologous Recombination in Vertebrate Cells Mol. Cell. Biol., August 1, 2003; 23(15): 5421 - 5430. [Abstract] [Full Text] [PDF]
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 S. Jin, H. Mao, R. W. Schnepp, S. M. Sykes, A. C. Silva, A. D. D'Andrea, and X. Hua Menin Associates with FANCD2, a Protein Involved in Repair of DNA Damage Cancer Res., July 15, 2003; 63(14): 4204 - 4210. [Abstract] [Full Text] [PDF]
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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]
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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]
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A. R. Meetei, S. Sechi, M. Wallisch, D. Yang, M. K. Young, H. Joenje, M. E. Hoatlin, and W. Wang A Multiprotein Nuclear Complex Connects Fanconi Anemia and Bloom Syndrome Mol. Cell. Biol., May 15, 2003; 23(10): 3417 - 3426. [Abstract] [Full Text] [PDF]
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 D. Sridharan, M. Brown, W. C. Lambert, L. W. McMahon, and M. W. Lambert Nonerythroid {alpha}II spectrin is required for recruitment of FANCA and XPF to nuclear foci induced by DNA interstrand cross-links J. Cell Sci., March 1, 2003; 116(5): 823 - 835. [Abstract] [Full Text] [PDF]
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D. I. Kutler, B. Singh, J. Satagopan, S. D. Batish, M. Berwick, P. F. Giampietro, H. Hanenberg, and A. D. Auerbach A 20-year perspective on the International Fanconi Anemia Registry (IFAR) Blood, February 15, 2003; 101(4): 1249 - 1256. [Abstract] [Full Text] [PDF]
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Q. Pang, T. A. Christianson, W. Keeble, T. Koretsky, and G. C. Bagby The Anti-apoptotic Function of Hsp70 in the Interferon-inducible Double-stranded RNA-dependent Protein Kinase-mediated Death Signaling Pathway Requires the Fanconi Anemia Protein, FANCC J. Biol. Chem., December 13, 2002; 277(51): 49638 - 49643. [Abstract] [Full Text] [PDF]
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D. Adachi, T. Oda, H. Yagasaki, K. Nakasato, T. Taniguchi, A. D. D'Andrea, S. Asano, and T. Yamashita Heterogeneous activation of the Fanconi anemia pathway by patient-derived FANCA mutants Hum. Mol. Genet., December 1, 2002; 11(25): 3125 - 3134. [Abstract] [Full Text] [PDF]
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H. Yagasaki, D. Adachi, T. Oda, I. Garcia-Higuera, N. Tetteh, A. D. D'Andrea, M. Futaki, S. Asano, and T. Yamashita A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA Blood, December 15, 2001; 98(13): 3650 - 3657. [Abstract] [Full Text] [PDF]
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 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]
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T. Otsuki, Y. Furukawa, K. Ikeda, H. Endo, T. Yamashita, A. Shinohara, A. Iwamatsu, K. Ozawa, and J. M. Liu Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex Hum. Mol. Genet., November 1, 2001; 10(23): 2651 - 2660. [Abstract] [Full Text] [PDF]
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 M. P. Wajnrajch, J. M. Gertner, Z. Huma, J. Popovic, K. Lin, P. C. Verlander, S. D. Batish, P. F. Giampietro, J. G. Davis, M. I. New, et al. Evaluation of Growth and Hormonal Status in Patients Referred to the International Fanconi Anemia Registry Pediatrics, April 1, 2001; 107(4): 744 - 754. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
and interferon (IFN)-
.
) or
corrected (+) with the FANCA cDNA (A, C), or FA-G (EUFA316)
lymphoblasts, which were uncorrected (
NLS).
Neither mutant conferred MMC resistance to the FA-A cells (data not
shown). Interestingly, only FANCA-H1110P was able to increase the FANCG protein levels (Figure 
-tubulin levels (cytoplasmic). Note that
the FANCA antiserum reacts nonspecifically with a protein that runs
just above the FANCA band (indicated by an arrow in the FANCA panel),
and the antiserum is present in all samples including the HSC72 cells,
which do not express any FANCA protein.
