Mutagenesis vol. 19 no. 3 pp. 237-244,
May 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Characterization of the hamster FancG/Xrcc9 gene and mutations in CHO UV40 and NM3
BBR Program, L441, Lawrence Livermore National Laboratory, PO Box 808, Livermore, CA 94550-0808, USA and 1School of Biological Sciences, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK
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The human FANCG/XRCC9 gene, which is defective in Fanconi anemia complementation group G (FA-G) cells, was first cloned by genetic complementation of the mitomycin C (MMC) sensitivity of CHO mutant UV40. The CHO NM3 mutant was subsequently assigned to the same complementation group. The parental AA8 CHO cells are hemizygous at the FancG locus, and we identified frameshift mutations that result in N-terminal truncations of the protein in both UV40 and NM3. Hypersensitivity to DNA cross-linking agents, such as MMC, typically characterizes FA cells. By introducing the native CHO FancG gene into mutant NM3, we demonstrate that hamster FancG fully corrects the 3-fold sensitivity to methyl methanesulfonate (MMS) as well as the 10-fold sensitivity to MMC, whereas resistance to ionizing radiation did not increase appreciably. In contrast, hamster cDNA transformants showed incomplete correction for both MMC and MMS sensitivity. The constitutively expressed FancG protein is present in the cytoplasmic, nuclear and chromatin fractions. FancG protein levels and subcellular localization do not change appreciably as a function of cell cycle position. Our results are consistent with roles of FancG in both the nuclear and cytoplasmic compartments to maintain genomic stability in response to various genotoxic agents.
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Introduction |
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Fanconi anemia (FA) is an autosomal recessive disorder that predisposes patients to bone marrow failure and/or a pronounced risk for cancer, predominantly myeloid leukemia. The disorder is genetically heterogeneous, involving at least eight different gene products. To date, at least seven of these genes (FANCA, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF and FANCG) have been cloned (Strathdee et al., 1992
; Lo Ten Foe et al., 1996; de Winter et al., 1998, 2000a,b; Timmers et al., 2001; Howlett et al., 2002), but the biochemical functions of their products remain largely unknown. Data suggesting that FANCB might be identical to BRCA2 was recently presented (Howlett et al., 2002). At the cellular level, FA cells are characterized by hypersensitivity to the cross-linking agents mitomycin C (MMC) and diepoxybutane (DEB) and an early study suggested that FA is defective in the repair of DNA crosslinks (Fujiwara et al., 1977). However, cross-sensitivity of FA cells to other DNA-damaging agents has not been thoroughly examined. Besides their sensitivity to cross-linking agents, FA cells often display sensitivity to growth in 20% O2, elevated chromosomal aberrations, alterations in cell cycle progression and response to growth factor levels, reduced viability accompanied by increased apoptosis and an increased sensitivity to the apoptotic effects of TNF
and interferon-
(reviewed in Buchwald and Moustacchi, 1998; Joenje and Patel, 2001; D’Andrea and Grompe, 2003). Moreover, data demonstrating partial co-localization of FANCD2 and BRCA1 proteins in ionizing radiation (IR)-induced foci (Garcia-Higuera et al., 2001) suggested a possible link between the FA protein ‘pathway’ involving a multiprotein complex (Garcia-Higuera et al., 1999; Waisfisz et al., 1999; de Winter et al., 2000c; Medhurst et al., 2001) and homologous recombination. The finding that FANCD1 is identical to BRCA2 (Howlett et al., 2002) further strengthened the link between FA and homologous recombination (Stewart and Elledge, 2002).
Human FANCG, cloned by complementation of FA-G lymphoblasts, proved to be identical to XRCC9 (de Winter et al., 1998), which was isolated by correcting the MMC sensitivity of the UV40 mutant of CHO cells (Liu et al., 1997). Isolated in a UV mutant screen (Busch et al., 1994), UV40 also displayed hypersensitivity to MMC (11-fold), ethyl methanesulfonate (EMS) (10-fold), methyl methanesulfonate (MMS) (5-fold) and -rays (
1.5-fold) relative to the parental AA8 cells. Furthermore, the defect causing UV sensitivity appeared to be unrelated to nucleotide excision repair, but rather involved an arrest of DNA replication in response to UV exposure (Busch et al., 1996). This defective recovery of DNA replication suggests an inability to coordinate lesion bypass or to initiate new replicons.
More recently, a second mutant CHO line, NM3, was assigned to the UV40 complementation group by its inability to complement UV40 in hybrid cells (Wilson et al., 2001). NM3 was isolated in a screen for mutants sensitive to another cross-linking agent, nitrogen mustard (Meyn et al., 1991), and showed a mutagen sensitivity profile remarkably similar to that of UV40, including UV, -ray, bleomycin and MMS sensitivity (Meyn et al., 1991; Wilson et al., 2001). Although both mutants were isolated from CHO AA8 cells, each is morphologically quite distinct from AA8 and from each other, suggesting adventitious genetic alterations acquired during their derivation.
An important difference between the CHO and human FANCG mutants is that FA-G lymphoblasts are not sensitive to UV, MMS or -rays (de Winter et al., 1998). There was the formal possibility that the primary mutation in UV40 might reside in another gene that is required for expression of FancG. (FancG is used to distinguish the hamster gene from human FANCG.) Therefore, we sequenced FancG from UV40 and NM3 cells and identified mutations in this gene. To further evaluate the role of FancG in the phenotype of these mutants, the hamster FancG gene was introduced into UV40 and NM3 cells. We found a clear difference in the level of complementation between hamster FancG cDNA and genomic clone transformants. Therefore, we also investigated whether the level of FancG protein level varies temporally and/or spatially during the cell cycle.
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Cell lines and culture conditions
The NM3 and UV40 mutants were derived from the CHO AA8 line (Meyn et al., 1991
; Busch et al., 1996). CHO and HeLa S3 cells were grown in -MEM medium containing 10% fetal bovine serum, 10 µg/ml penicillin and 10 U/ml streptomycin. UV40 cultures were maintained in suspension in roller tubes revolving at 40 r.p.m. and NM3 cultures were grown as monolayers. 40cX9.31 is a cDNA transformant of UV40 that expresses human FANCG from a CMV viral promoter (Liu et al., 1997).
Cloning of the hamster FancG gene
The full-length hamster FancG open reading frame (ORF) was determined by 5'- and 3'-RACE and cloned into the mammalian expression vector pCMV-Tag (Stratagene, La Jolla, CA) in-frame with an N-terminal Flag epitope. The integrity of the Flag–FancG fusion construct (cXR9.Flag) was confirmed by sequencing and in vitro translation. The full-length CHO FancG ORF was used to probe filters containing an arrayed BAC library constructed from AA8 genomic DNA (obtained from Pieter de Jong, Roswell Park Cancer Center). Positive clones were re-screened by PCR with primers specific to the 5'- and 3'-ends of FancG. BAC clone 174 was chosen for further study.
FancG complementation of UV40 and NM3 cells
The cXR9.Flag construct was introduced into NM3 and UV40 cells using Lipofectamine 2000 (Gibco BRL) or by electroporation, respectively, as previously described (Liu et al., 1997). After 48 h, transfected cells were seeded into 100 mm dishes at 1 x 105 per dish, and treated with 1 mg/ml Geneticin (Sigma) and 70 (for UV40) or 200 (for NM3) nM MMC for 7–14 days. Twelve clones of each cell line were picked into a 12-well tray, treated with 0.5 mg/ml Geneticin (G418) plus 100 nM MMC for 10 days and evaluated in a cytotoxicity assay (Hoy et al., 1984). For gene transfection, 5 µg hamster BAC clone 174 was electroporated into UV40 or NM3 cells (Liu et al., 1997). Since no positive selection marker was present on the BAC vector, transformants were selected in the presence of MMC for 10 days as described above. No transformants were obtained for UV40 in three experiments. Twelve transformants in NM3 were grown for 5 days in a 12-well dish containing 200 nM MMC and expanded into non-selective medium. Two clones with the most robust growth (NM3.g1 and NM3.g10) were chosen for further studies.
Cytotoxicity and survival experiments
For each cell line, 1 x 104 cells/well were seeded into a 12-well dish in 2 ml of -MEM. Increasing concentrations of MMC were prepared in -MEM, starting at a concentration of 100 nM and increasing by factors of 1.3 up to the highest concentration of 1.1 mM. The first well served as the untreated control, and cells were incubated at 37°C in 6% CO2 for 5–7 days until the untreated wells reached confluence. Cells were then fixed and stained with crystal violet as previously described (Hoy et al., 1984). Since UV40 attaches very poorly to plastic, survival experiments were performed only with NM3 and its FancG-complemented derivatives. NM3 does not grow well in suspension, so all cell lines were treated in monolayer. Aliquots of 5 x 106 cells were treated with MMC or MMS [stocks prepared in phosphate-buffered saline (PBS)] in a volume of 10 ml in -MEM for 1 h at 37°C. The cells were then rinsed in room temperature PBS, trypsinized, counted and plated in triplicate dishes for each dose. Dishes were incubated for 8–10 (for AA8) or 10–13 (for NM3 and its derivatives) days before fixing and staining. Each data point represents an average of at least two independent experiments.
Cell synchronization and analysis
Approximately 2 x 108 exponentially growing HeLa-S3 cells were synchronized by size using centrifugal elutriation in a J6 MI centrifuge (Beckman Coulter) and at least 15 size-separated fractions were collected. For each fraction, 5 x 105 cells were pulse labeled with 10 µM bromodeoxyuridine (BrdUrd) for 30 min in roller-drum cultures. DNA profiles were obtained by staining nuclei with propidium iodide for DNA content and FITC-conjugated anti-BrdUrd antibody (BD Biosciences) for DNA synthesis following methods as described (Kastan et al., 1991). Labeled nuclei were analyzed on a FACScan flow cytometer (BD Biosciences) to determine the fraction of cells in the G1, S and G2 phases.
Mutation analysis
Genomic PCR was performed to amplify the FancG gene from mutants UV40 and NM3 using three sets of primer pairs which produced partially overlapping fragments of 5.5, 2.3 and 1 kb that spanned most of the gene. PCR was performed with a Genomic PCR kit from Clontech using 100 ng genomic DNA. PCR products were sheared by sonication, subcloned into vector M13 and sequenced to 8-fold redundancy. Sequence reads were assembled with PHRAP. The resulting consensus sequence was compared with that of the hamster FancG cDNA using BLAST (Altschul et al., 1990). Regions containing putative mutations in UV40 and NM3 were amplified from genomic DNA in triplicate reactions and the PCR products were sequenced directly to confirm the mutations.
Northern analysis
Total RNA was isolated from 6 x 107 cells and subsequent purification was performed using an oligo(dT)-based magnetic bead purification scheme (Promega) following the manufacturer’s protocol. An aliquot of 5 µg of mRNA from each cell line was loaded onto a 1% formaldehyde–agarose gel and subjected to electrophoresis for 4 h in 1x MOPS running buffer, with one exchange of buffer midway through the run. Samples were transferred to nylon membrane (Ambion) for 1.5 h and UV-cross-linked. Hybridization with a radiolabeled probe representing the entire FancG ORF was performed at 68°C overnight in ExpressHyb buffer (Clontech). Washes were performed as recommended by the manufacturer. The blot was exposed to a phosphor screen for up to 4 days and the image processed on a Molecular Dynamics Phosphoimager.
FANCG antibody purification
Five milligrams of gel-purified His6–FANCG protein (N-terminal His fusion) expressed in Escherichia coli was used to immunize two rabbits (HTI Bioproducts Inc., Ramona, CA). To produce a FANCG affinity column for antibody purification, 1 mg insoluble His6–FANCG protein (C-terminal His fusion) was dialyzed at room temperature into 1 l of 0.1% SDS, 0.5 M NaCl and 0.1 M sodium bicarbonate, pH 8.4, followed by dialysis in 1 l of the same buffer without SDS. The His6–FANCG protein was cross-linked to CNBr-activated Sepharose (Amersham-Pharmacia Biotech) by mixing equal volumes of resin and protein (1 ml each) together at room temperature for 5 h. After protein coupling, excess reactive groups were blocked by treating the His6–FANCG protein-linked Sepharose with 5 ml of 100 mM Tris–HCl, pH 8.0, at room temperature for 2 h. To purify the FANCG antibody, 10 ml of whole serum was recirculated five times across the His6–FANCG column equilibrated in PBS (pH 7.2). The column was washed with 10 column volumes each of PBS and double strength PBS containing 0.05% Tween-20 and then with 3 column volumes of PBS. The FANCG antibody was eluted using 15 column volumes of 0.1 M diethylamine. Fractions (1 ml) were collected into tubes containing 0.5 ml of 0.5 M potassium MES, pH 6.0, to neutralize the diethylamine. The antibody peak was located by measuring the OD280 of each fraction. Peak fractions were pooled and dialyzed overnight at 4°C in 1 l of PBS. After dialysis, precipitated protein was removed by centrifugation. The antibody supernatant was concentrated to an OD280 of 3.0 using a Centricon-30 (Millipore) and stored at –80°C in aliquots.
Cell lysis and western blotting
Nuclear and cytoplasmic extracts were prepared from elutriated fractions after samples were taken for FACS analysis (5 x 106 cells), using a NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce). The remaining pellet material containing chromatin was washed twice with buffer A (10 mM HEPES, pH 8, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol and 1 mM DTT), resuspended in Laemmelli buffer and sonicated briefly. A western blot with anti-histone H3 antibody was conducted to confirm that the pellet material contained chromatin-associated proteins (data not shown). Extracts were quantitated using a Coomassie Protein Assay kit (Pierce) and normalized before loading. To confirm that loading was equal for all samples, gels were stained with GelCode reagent (Pierce) after transfer. Western blotting was conducted following standard procedures with ECL detection (Amersham Biosciences), using 5% non-fat dried milk as a blocker, with the following dilutions: 1:2000 of anti-FANCG primary antibody, 1:500 of anti-actin primary antibody and 1:1000 of the appropriate secondary antibodies. Exposed films were scanned and the band intensities were determined using ImageQuant phosphoimager software (Molecular Dynamics).
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Cloning of the hamster FancG gene
Previous complementation of UV40 with human FANCG/XRCC9 cDNA resulted in only partial correction of the mutant’s sensitivity to DNA-damaging agents (Liu et al., 1997
). To assess whether this result was due to heterology between human and hamster FANCG, we cloned the CHO FancG gene. This isolation was done by first performing RT–PCR with primers derived from regions conserved between human FANCG and a mouse EST (IMAGE clone 425878) found in dbEST. A single round each of 5'- and 3'-RACE resulted in a reconstructed transcript of 2.3 kb, which agrees with that seen by northern blot analysis of AA8 cells (Liu et al., 1997) (Figure 4). The translated sequence of the CHO FancG gene predicts a leucine-rich protein of 621 amino acids with a corresponding molecular weight of 68.1 kDa and a predicted pI of 5.5. A comparison of the predicted protein with the human and mouse orthologs is shown in Figure 1.
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The human and rodent FANCG proteins share 70% identity, which is somewhat low compared with the values for several DNA repair proteins, e.g. XRCC1 (86%) (Brookman et al., 1994
), XRCC2 (78%) (Cartwright et al., 1998) and XRCC5/Ku86 (76%) (Errami et al., 1996). This level of conservation is consistent with that observed between the human and murine FANCA and FANCC proteins, which are 65 and 68% identical, respectively (Wevrick et al., 1993; Wong et al., 2000). Recently, seven tetratricopeptide repeat motifs (TPRs) covering a major part of human FancG protein were identified by sequence comparisons with FancG from two species of fish, Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) (Blom et al., 2004). TPRs are degenerate 34 amino acid repeat motifs that act as scaffolds to mediate protein–protein interactions, often in multiprotein complexes.
Secondary structure predictions suggest that the protein has a high -helix content, and presumptive leucine zippers have been identified in the human and rodent sequences, although the exact locations of this motif differ between the human and rodent homologs (see Figure 1). No nuclear localization signals were identified, but analysis with the program PSORT (Nakai and Kanehisa, 1992) suggests that human FANCG would localize primarily in the nuclear and mitochondrial subcellular compartments. Human FANCG was recently reported to localize to both the cytoplasm and the nucleus as a function of the presence and amount of FancA protein in those subcellular compartments (Kruyt et al., 1999), consistent with our results (see Figure 6).
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Full complementation of MMC and MMS sensitivity of mutant NM3 by hamster FancG
The CHO FancG cDNA was cloned as an N-terminal Flag epitope fusion into the pCMV-Tag vector (Stratagene, La Jolla, CA) to facilitate detection of the FancG protein in cell extracts. The integrity of the fusion construct was confirmed by sequencing and in vitro translation (data not shown). Transfection of this plasmid into UV40 cells yielded transformants that were resistant to both G418 and MMC at a frequency of 2 x 10–4, and two transformants (40.Flag2 and 40.Flag6) were selected for further study on the basis of their improved growth rates relative to UV40. The two transformants were characterized for their resistance to MMC in a 12-well differential cytotoxicity assay (Hoy et al., 1984
), with wild-type AA8 and a human FANCG cDNA transformant [40cXR9.31 (Liu et al., 1997)] as controls. As shown in Table I, the two hamster cDNA transformants were no more resistant to MMC than the human cDNA transformant, which had 50% of the wild-type level of resistance. Transformants 40.Flag2 and 40.Flag6 did exhibit slightly shorter doubling times of 18–20 h, relative to UV40 and 40cXR9.31. The incomplete correction by the hamster cDNA suggests that inappropriate expression could be detrimental to normal function. In addition, 40.Flag2 and 40.Flag6 also exhibited the same elevated level of spontaneous sister chromatid exchange observed in UV40 cells (data not shown), suggesting that this trait is unrelated to FancG.
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We hypothesized that the incomplete correction of UV40 by FancG cDNA was related to abnormal expression and that the hamster gene would provide full complementation. An AA8-derived BAC genomic clone from an arrayed library was identified by hybridization with the hamster FancG cDNA. PCR primers targeted to the 5'- and 3'-ends of the ORF were employed to verify that the entire gene was present in the genomic clone. UV40 and NM3 cells were electroporated with 5 µg BAC (clone 174) DNA and grown for 10 days in medium containing 70 or 200 nM MMC, respectively. No transformants were obtained in the UV40 cell line in three independent experiments (for reasons we have not established), but many robust transformants were obtained with NM3. Differential cytotoxicity assays performed on two transformants (NM3.g1 and NM3.g10) suggested full restoration of MMC resistance relative to AA8 cells (data not shown).
To accurately compare MMC resistance in cDNA and genomic transformants of NM3, we performed survival experiments for colony-forming ability (Figure 2A). 40.Flag2 and 40.Flag6 were slightly less resistant than AA8 whereas NM3.g1 and NM3.g10 were, on average, slightly more resistant than AA8. The cDNA transformants were 1.6-fold more sensitive to MMC than wild-type AA8 at the D37 dose. Although FA lymphoblasts are generally not significantly sensitive to MMS (de Winter et al., 1998; Carreau et al., 1999), both UV40 and NM3 were reported to exhibit pronounced MMS sensitivity, i.e. 5- to 8-fold (Meyn et al., 1991; Busch et al., 1996; Wilson et al., 2001). Therefore, we tested whether the CHO FancG gene corrected the MMS sensitivity of NM3 cells. Indeed, the genomic transformants were fully corrected while the cDNA transformants had intermediate resistance (Figure 2B).
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The NM3 and UV40 mutants were originally reported to be sensitive to IR (Meyn et al., 1991
) by a factor of 1.5 in terms of dose reduction (Busch et al., 1996). However, we found little sensitivity for NM3 and little or no increase in IR resistance in the two BAC-complemented NM3 clones (Figure 2C).
Frameshift mutations underlying loss of FancG protein in CHO mutants
To test the possibility that FancG might be a phenotypic suppressor gene conferring correction to transfected UV40 and NM3 mutants, we identified the causative mutations in both mutants. We determined the genomic structure of the human FANCG locus by sequencing a human P1 clone (accession no. AC004472; our unpublished data). This information was used to identify primer pairs for amplifying CHO FancG, resulting in partially overlapping genomic fragments (5.5, 2.3 and 1 kb) that spanned most of the gene.
Since both UV40 and NM3 were isolated after mutagenesis of AA8 cells with the mutagen ICR171 (Meyn et al., 1991), we expected to observe frameshift mutations. As shown in Figure 3A, mutations causing premature truncation of FancG were identified in both mutants: in exon 1 of UV40 and exon 3 of NM3. In UV40, six out-of-frame residues would be added after the ninth amino acid (leucine) prior to termination, while for NM3, alanine 92 is changed to a glycine, followed by a termination codon. Since only one mutation in FancG was identified in each cell line, we tested whether the parental AA8 cells are hemizygous at this locus. We detected a single copy of the FancG gene by FISH analysis in AA8 metaphase chromosomes (Figure 3B). This finding of hemizygosity helps to explain the isolation of the two independent mutants, UV40 and NM3, in the quasi-diploid AA8 cells.
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The mutation in UV40 cells was hypothesized to destabilize the FancG mRNA, as no transcript was detected previously by northern analysis (Liu et al., 1997
). Given that the cDNA transformants were only partially corrected for the end-points assayed, we wished to determine transcript levels in the cDNA and genomic transformants of NM3. As shown in Figure 4A, FancG transcript is present at approximately the wild-type level in the genomic transformants (NM3.g1 and NM3.g10) and in UV40. NM3 cells appear to express a lower level of transcript relative to UV40 and AA8. Surprisingly, expression of the FANCG transcript in the cDNA transformants (in both UV40 and NM3 backgrounds) is 10-fold higher than the wild-type level (Figure 4A, lanes 6–9). Flag-tagged FancG mRNA migrates as a 2.6 kb band due to the additional N-terminal Flag tag sequence derived from the pCMV vector.
The presence of FancG transcript in the UV40 mutant differed from previous results (Liu et al., 1997). To confirm that transcript is present in these mutant cell lines, we performed RT–PCR on the same mRNA samples. As shown in Figure 4C, primers specific to the last 979 bp of FancG cDNA amplified a fragment of the expected size in both mutant cell lines, as well as in AA8. Since these primers span exons 8–14, genomic contamination would result in an 4 kb fragment, which was not observed. It seems likely that the hamster cDNA used here is a more sensitive probe than the human cDNA fragment used previously (Liu et al., 1997). It is also possible that the stability of the mRNA could change over time, depending on culture conditions.
To determine whether the high FancG transcript levels in the cDNA transformants resulted in excess FancG protein, western blots were performed with whole cell lysates, using a polyclonal antibody raised against human FANCG. As shown in Figure 5, an 64 kDa protein corresponding to CHO FancG is seen in AA8 (lanes 1 and 7), but not in NM3 extract (lane 2), confirming that the frameshift mutation in NM3 eliminates the protein. Notably, both genomic and cDNA transformants had FancG levels similar to that of AA8 cells, indicating that protein level alone does not account for the incomplete correction in cDNA transformants.
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Constancy of FancG levels and compartmentalization during the cell cycle
The human FANCG protein is known to physically associate with FANCA and FANCC, as shown by co-immunoprecipitation (Garcia-Higuera et al., 1999
; Kruyt et al., 1999; Waisfisz et al., 1999), and with FANCF, as shown by the yeast 2-hybrid system (Gordon and Buchwald, 2003). Although the FANCA and FANCC transcripts appear to be constitutively expressed (Heinrich et al., 2000), FANCC protein level changes during the cell cycle and FANCC protein physically interacts with Cdc2 (Kupfer et al., 1997). Given that cDNA transformants were only partially corrected, we wished to assess whether FancG is regulated temporally and/or spatially in CHO cells.
AA8 and HeLa S3 cells were synchronized by centrifugal elutriation and the fractions were analyzed by western blotting. Using an antibody made against human FancG, the sensitivity of detection of hamster FancG was mediocre, but the protein level did not appear to change during the cell cycle (results not shown). Figure 6 shows the results for HeLa S3 cells, which provided clearer results. The synchronous fractions ranged from almost all cells in G1 (fraction 1) to most of the cells in S (fractions 5 and 6) to most of the cells in G2/M (fraction 7). The samples were analyzed for cytoplasmic, nuclear and chromatin fractions. The level of human FANCG in each fraction did not appear to change through the cell cycle, with the possible exception that the nuclear content is low in the first two cell fractions containing mostly G1 cells. The two bands in the cytoplasmic fraction indicate that there is a post-translational modification of the protein. Phosphorylated forms of FANCG have been reported in transformed human cells (Futaki et al., 2001; Qiao et al., 2001).
Lack of damage inducibility of FancG protein
Given that NM3 and UV40 exhibit sensitivity to a broad spectrum of DNA-damaging agents, we wished to determine whether the FancG protein is induced or stabilized in response to DNA damage. AA8 cells were treated with a dose of MMC that results in 30% survival, harvested at several time points after treatment and analyzed by western blot. No significant change in FancG protein was seen at any time and no change occurred in subcellular localization (data not shown). Our findings for FancG are consistent with the published lack of inducibility of FANCC protein in response to treatment with MMC, DEB, hydrogen peroxide and -irradiation (Tower et al., 1998).
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FA cells have been characterized as having sensitivity primarily to the DNA interstrand cross-linking agents MMC and DEB, and cross-sensitivity to other genotoxic agents is not typically associated with these cells in the literature. However, several studies show a modest sensitivity to IR with FA lymphocytes and fibroblasts (Bigelow et al., 1979
; Deschavanne et al., 1986) and splenic lymphocytes in FancG knockout mice (Yang et al., 2001). Our results clearly show that the FancG protein in CHO cells confers protection against killing by the methylating agent MMS. Therefore, we compared the MMS sensitivity of FA-G EUFA143 lymphoblasts (de Winter et al., 1998) with FANCG-complemented transformants (using the human gene, not cDNA) that are fully corrected for MMC sensitivity; we observed little or no difference in MMS sensitivity between the isogenic cell lines (unpublished data). Thus, the phenotypic differences observed between CHO and FA lymphoblasts may represent cell lineage-specific differences, or possibly species differences.
Introduction of the FancG gene into NM3 cells conferred wild-type levels of resistance to MMC and MMS, whereas the cDNA under the control of a strong CMV promoter only partially complemented. The high transcript levels seen in cDNA transformants compared with genomic transformants were not associated with increased FancG on western blots. This difference might be explained by instability of excess FancG that is not complexed with FancA and other FA proteins (Garcia-Higuera et al., 2000). Furthermore, nucleotide sequence analysis of the cgXR9.Flag construct eliminated the possibility that a mutant protein was being expressed, as the correct FancG sequence was obtained. The basis of the incomplete complementation seen with the FancG cDNA construct is unclear, but may be attributable to the Flag epitope, which could impair the function of the protein. Curiously, we found that a Flag antibody did not detect the epitope in extracts of the NM3 Flag transformants. Western blotting for FancG detected a faint higher molecular weight band specifically in the two NM3 Flag transformants. We speculate that there may be modified isoforms of FancG, generated as a consequence of having the extra tag and linker sequences in the cDNA construct, and that this isoform may interfere with normal FancG activity, contributing to the reduced correction in the cDNA transformants. However, this issue would require more definitive analysis for clarification.
In contrast to FANCC, which was reported to increase several fold during S phase in thymidine-synchronized HeLa cells (Kupfer et al., 1997), we found that FancG remained approximately constant in both the cytoplasm and nucleus (Figure 6). Evidence for cytoplasmic functions of FANCC in redox metabolism has been presented (Kruyt et al., 1998; Cumming et al., 2001), which led to intense debate (Cumming and Buchwald, 2001; D’Andrea, 2001). Human EUFA143 FA-G lymphoblasts, when compared with a cDNA-complemented transformant, were reported to have an increased level of CYP2E1 (Futaki et al., 2002). In the complemented cells, CYP2E1 was found to interact with FANCG (Futaki et al., 2002). This study also reported that FA-G cells treated with hydrogen peroxide or MMC had elevated oxidative DNA damage (7,8-dihydro-8-oxoguanine) when compared with FANCG-complemented cells. The authors suggested that the interaction of FANCG with CYP2E1 might alter redox metabolism and increase DNA oxidation. In the nucleus, FANCG is a member of the core complex containing the A, C, E, F, G and L proteins (D’Andrea and Grompe, 2003; Meetei et al., 2003) that are required for mono-ubiquitination of FANCD2 (Gregory et al., 2003) and resistance to genotoxic agents. Moreover, FANCG is reported to interact directly with BRCA2 (Hussain et al., 2003). Thus, it is of interest to identify mutations that separate the nuclear and putative cytoplasmic functions of FANCG.
The literature on the biochemical functions of the FA proteins remains complex and often seemingly contradictory. For example, a recent study concludes that normal diploid fibroblasts of FA complementation groups A, C, D2 and G are defective in end joining repair of double-strand breaks in transfected plasmids and are hypersensitive to killing by transfected restriction enzymes (Donahue and Campbell, 2002). These results would suggest an FA phenotype of sensitivity to IR, which is only reported sporadically. We found only a slight, if any, increase in IR resistance in BAC-complemented NM3 cells (Figure 2C). Moreover, the FancG knockout mutant of chicken DT40 cells is not IR sensitive for colony formation (although there is some sensitivity for chromosomal aberrations in G2 cells) (Yamamoto et al., 2003). To develop an isogenic system, we recently constructed a CHO FancG knockout mutant (Tebbs et al., 2002), whose characterization is expected to provide further insight into FancG function.
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Acknowledgements |
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We thank Jennifer Dias, Marilyn Ramsey, Kathryn Segalle and Vijay Viswanathan for technical assistance. This work was performed at the LLNL under the auspices of the US Department of Energy under contract no. W-7405-ENG-48. The research was funded by the Low Dose Radiation Research Program, Biological and Environmental Research (BER), US Department of Energy (project ‘Assessing biological function of DNA damage response genes’; http://lowdose.tricity.wsu.edu/current_fund.htm) and by NIH/NCI grant CA89405. A portion of this work (N.J.J.) was funded by the North West Cancer Research Fund in the UK.
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2To whom correspondence should be addressed. Tel: +1 925 422 5658; Fax: +1 925 422 2099; Email: thompson14{at}llnl.gov
This publication is dedicated to the memory and scientific contributions of Dr David B.Busch, who isolated the UV40 mutant in his large-scale UV mutant screens. David developed leukemia and passed away in April 2002. |
References |
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