Oxidative stress/damage induces multimerization and interaction of Fanconi anemia proteins

Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, C, E, F, G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomer under non-oxidizing conditions, while they become multimers following H 2 O 2 treatment. Treatment of cells with oxidizing agent not only triggers multimeric complex of FANCA and FANCG in vivo , but also induces the interaction between FANCA and FANCG. N-ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro . Together, we conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming FANC complex and their function.


INTRODUCTION
Fanconi anemia (FANC) is an autosomal recessive disorder characterized by chromosomal instability and defective DNA repair, and FANC-deficient cells exhibit extreme sensitivity towards oxygen and DNA crosslinking agents such as diepoxybutane and mitomycin C (1-3).
Mutations in any of eight different genes lead to FANC disease, a degree of genetic heterogeneity comparable to that of other DNA repair disorders, suggesting that each group represents a distinct protein.
FANCA and FANCG proteins are part of a large nuclear protein complex required for their function, and the disruption of this complex results in the specific cellular and clinical phenotype common to most FANC complementation groups (4). FANCA gene encodes a 162-kDa phosphoprotein and its phosphorylation correlated with FANCA/FANCC protein accumulation in the nucleus (5). FANCA mutant cells isolated from a FANC patient was defective in its phosphorylation and failed to bind to FANCC. Furthermore, a mutant FANCA protein failed to complement the MMC sensitivity of FANCA-/-cells, suggesting that FANCA phosphorylation may be involved in FANCC interaction, nuclear localization of FANCA, or its function in crosslink repair. FANCG gene encodes a 65-kDa protein and was identified as human XRCC9.
XRCC-9 (FANCG) complements the Chinese Hamster Ovary (CHO) mutant UV-40 cell line that is hypersensitive to UV, ionizing radiation, simple alkylating agents, and DNA cross-linking agents (6,7). The mutant cells also show a high level of spontaneous chromosomal aberrations that can be fully corrected by introduction of XRCC9 cDNA transformants (7). A possible by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 4 involvement of FANC proteins in DNA repair was strengthened by recent findings on the interaction of FANCD1 with BRCA1 following DNA damage (8). FANCD1 is identical to BRCA2 gene and is unique among FANC genes in that it is essential for the formation of Rad51 foci in response to ionizing radiation (9), suggesting that it may be involved in homologous recombination-mediated strand break repair.

Cloning of FANCA and FANCG genes
The cDNA for human FANCG was subcloned into pcDNA6.

Protein Purification
Anti-FLAG M2 agarose was used in purifying Flag-FANCA protein and used according to the manufacturer's (Sigma) instructions. The supernatants from transfected COS cell lysates were incubated at 4°C for at least 4 h with anti-FLAG M2 affinity gel (Sigma) that had been preequilibrated in PBS buffer. The beads were then washed three times with 1% NP-40 in PBS buffer. In the final step, the M2 column was eluted with 3 column volumes of 500 µg/ml FLAG 7 peptide. GST-FANCG proteins were expressed in Sf9 insect cells following recombinant baculovirus infection (19) and purified by glutathione-Sepharose resin (Amersham Biosciences) and by FPLC on a 1-ml HiTrap Q column (Amersham Biosciences) (20).

SDS-PAGE and Western blot analysis
Cell lysates (25 µg per lane) or purified proteins were were resolved on 6 to 8% SDSpolyacrylamide gels under reducing conditions (10 mM DTT or 1% β-mercaptoethanol) or under non-reducing conditions. Proteins were then transferred to polyvinylidene difluoride membrane, probed with an anti-FANCA or -FANCG antibody (a rabbit polyclonal IgG), -FANCD2 antibody (monoclonal mouse IgG, Novus) followed by horseradish peroxidaseconjugated secondary antibody. Proteins were visualized by using the ECL system (Amersham Biosciences).

Immunoprecipitation
Cell lysates were clarified by centrifugation in a microcentrifuge for 10 minutes at 8,000 x g at 4 °C. The supernatants were removed and incubated with ANTI-FLAG ® M2 antibody for 8

Multimerization of FANC proteins under oxidizing conditions
Increasing evidence points to a role for FANC proteins in redox signaling and the repair of oxidative damages (22)(23)(24). As with this, cells lacking FANC genes showed hypersensitivity to H 2 O 2 treatment (13; data not shown). Since several FANC proteins (FANCA, FANCC, and FANCG) contain unusually high number of cysteines, we wondered whether some of these residues were directly involved in redox regulation. To examine for redox regulation of FANC proteins, insect cells were transfected with recombinant baculovirus expressing GST-FANCG.
Purified FANCG protein was analyzed on SDS-PAGE following a brief air oxidation and addition of reducing agent. Purified GST-FANCG migrated as multimers (>250-kDa in size) under oxidizing condition, while it migrated as a monomer with increasing amount of DTT ( Figure 1A). Transition from monomer to multimer likely occurred through the formation of intermolecular disulfide linkage(s) on FANCG not the GST portion of the fusion protein since purified GST did not form multimers regardless of its redox status ( Figure 1B). It is interesting to note that GST-FANCG (90-kDa) migrated more than one form of multimers in a range from 180-kDa to a much larger in size under oxidizing conditions ( Figure 1A & 1C).

COS cells
We next examined whether a redox potential affects monomeric/multimeric change of FANC proteins. COS cells were transfected with either a mammalian vector encoding FANCA, FANCG, or cotransfected with both FANCA and FANCG vectors. Cell lysates were examined for expression of FANCA and FANCG by Western blotting. A stable expression of FANCA was observed regardless of the presence of FANCG (Figure 2A lanes 1 & 2). On the other hand, 9 FANCG protein was stably expressed only in the presence of FANCA and was hardly detected without FANCA (Figure 2A, lane 3 vs lane 4), suggesting that FANCA is necessary for stable expression of FANCG in mammalian cells (25). Similar to an in vitro study with purified GST-FANCG ( Figure 1A), FANCA multimers were converted to a monomer following incubation with increasing amount of DTT ( Figure 2B), suggesting that multimerization of FANCA occurs through intermolecular disulfide linkage(s). FANCA and FANCG in COS cells exist as monomers under reduced condition (lane 1, Figure 2C), but converted to various sizes of multimer(s) under non-reducing condition (lane 2, Figure 2C).  (28,29). On the other hand, intermolecular disulfide linkage(s) also attributes the control of protein function through oxidation state of conserved cysteines. Glutathione forms a dimer via disulfide linkage upon redox change and is an important cellular defense against oxidative stress (30). Fos/Jun (31,32) and NF-Y complex (33) are the transcription factors that form heterodimer for their function in 12 response to oxidative stress. It should be pointed out however some of the complexes involving disulfide linkage(s) such as gp70-Pr15E interaction may not be regulated by redox change since the complexes are resistant to treatment with reducing agent(s) (34). Given the fact that redox change facilitates physical interaction of FANCA with FANCG, the FANCA-FANCG interaction is likely controlled by intermolecular disulfide linkage(s). Identification of cysteine sites on FANCA and FANCG would be necessary to clarify the roles for the FANCA-FANCG complex in damage signaling and DNA repair.

Multimerization of FANC proteins or the FANCA-FANCG interaction is induced by either H 2 O 2
or MMC treatment. H 2 O 2 not only oxidizes protein, but also directly attacks DNA to oxidize bases on chromosomal DNA; the latter is usually repaired by base excision repair (22,35).
MMC however mostly induces inter-and intrastrand DNA crosslinks, although it can cause oxidative damage on non-DNA components. Resistance to MMC was shown to require direct interaction between FANCA and FANCG in the nucleus (15). In addition, cytoplasmic localization of FANCC is important for correction of MMC sensitivity in the FANCC phenotype, suggesting that MMC sensitivity may also be coupled to cytoplasmic defense mechanism against oxidative damage (36). It is not clear however whether multimerization of FANC proteins or the FANCA-FANCG interaction is limited to certain oxidative DNA damage(s), or a response to various DNA damaging agents. possibility exists that the entire FANC complex may occur through disulfide linkage(s) following redox change. Functional implication of FANC multimers in response to oxidative stress is not clear, however, the earlier study suggested that FANCA dimerization is required for formation of FANC complex (18). It is possible that multimers of FANCG compared to the FANCG monomer preferentially interacts with FANCA ( Figure 5B). Alternatively, multimerization of the FANC proteins has a unique role following oxidative damage that is different from those for the FANC complex.
FANCA-FANCG interaction is not only crucial for stability of FANCG (25; Figure 2A), but also essential for their function in the nucleus. FANCA-FANCG interaction concomitantly occurred with monoubiquitination of FANCD2 following oxidative damage (data not shown). Previous study showed that MMC treatment induced the FANC proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FANCD2 cells but not in mutant FANCA cells (37). Cells lacking FANCA compared to wild-type cells showed a significantly lower level of FANCL (38). FANCL with its E3 ubiquitin ligase activity is likely a catalytic subunit required for monoubiquitination of FANCD2 (38). FANCA may play an essential role in monoubiquitination of FANCD2 via stabilization of FANCL and its E3 ubiquitin ligase activity in the nucleus. Both FANCA and FANCD2 are phosphoproteins and their phosphorylation appears to be induced by DNA damage (5,39). It would be interesting to see whether the FANCA phosphorylation also affects interaction between FANCA and FANCG.
FANCA and FANCG, once forming a complex, likely serve as a core to facilitate the formation of a larger FANC complex containing at least five FANC proteins (A, C, E, F, and G) following oxidative damage ( Figure 6). FANC complex stabilizes FANCL (formally known as PHF9) (38), a new 14 member of FANC essential for monoubiquitination and redistribution of FANCD2 to nuclear foci containing BRCA1 following DNA damage (38). FANC complex also interacts with BRCA2 (FANCD1), a protein involved in recombinational repair pathway through regulation of Rad51 activity (9,40,41). In addition, FANC complex is associated with Bloom Syndrome complex, a large complex containing RecQ helicase, replication protein A, and topoisomerase III (42). Further studies would be necessary to see whether the FANCA-FANCG interaction via disulfide linkage(s) is essential for functions of the FANC complex in damage-induced cell cycle checkpoint and DNA repair described above ( Figure 6).