Fanconi anaemia proteins: major roles in cell protection against oxidative damage

Fanconi anaemia (FA) is a cancer-prone genetic disorder that is characterised by cytogenetic instability and redox abnormalities. Although rare subtypes of FA (B, D1 and D2) have been implicated in DNA repair through links with BRCA1 and BRCA2, such a role has yet to be demonstrated for gene products of the common subtypes. Instead, these products have been strongly implicated in xenobiotic metabolism and redox homeostasis through interactions of FANCC with cytochrome P-450 reductase and with glutathione S-transferase, and of FANCG with cytochrome P-450 2E1, as well as redox-dependent signalling through an interaction between FANCA and Akt kinase. We hypothesise that FA proteins act directly (via FANCC and FANCG) and indirectly (via FANCA, BRCA2 and FANCD2) with the machinery of cellular defence to modulate oxidative stress. The latter interactions may co-ordinate the link between the response to DNA damage and oxidative stress parameters (3, 6-12).     

Fanconi anemia: genes and function(s) revisited

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.     

New Advances in the DNA Damage Response Network of Fanconi Anemia and BRCA proteins: FAAP95 Replaces BRCA2 as the True FANCB Protein

Fanconi anemia (FA) proteins function in a DNA damage response pathway that appears to be part of the network including breast cancer susceptibility gene products, BRCA1 and BRCA2. In response to DNA damage or replication signals, a nuclear FA core complex of at least 6 FA proteins (FANCA, FANCC, FANCE, FANCF, FANCG, and FANCL) is activated and leads to monoubiquitination of the downstream FA protein, FANCD2. One puzzling question for this pathway is the role of BRCA2. A previous study has proposed that BRCA2 could be identical to two FA proteins: FANCD1, which functions either downstream or in a parallel pathway; and FANCB, which functions upstream of the FANCD2 monoubiquitination. Now, a new study shows that the real FANCB protein is not BRCA2, but a previously uncharacterized component of the FA core complex, FAAP95, suggesting that BRCA2 does not act upstream of the FA pathway. Interestingly, the newly discovered FANCB gene is X-linked and subject to X-inactivation. The presence of a single active copy of FANCB and its essentiality for a functional FA-BRCA pathway make it a potentially vulnerable component of the cellular machinery that maintains genomic integrity.     

FANCD1/BRCA2 plays predominant role in the repair of DNA damage induced by ACNU or TMZ

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.     

Crosstalk between BRCA‐Fanconi anemia and mismatch repair pathways prevents MSH2‐dependent aberrant DNA damage responses

Several proteins in the BRCA‐Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA‐FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA‐FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18‐dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2‐null primary mouse cells. Thus, we propose that regulation of MSH2‐dependent DNA damage response underlies the importance of interactions between BRCA‐FA and MMR pathways.     

Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiquitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation-induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.     

An ATR- and BRCA1-mediated Fanconi anemia pathway is required for activating the G2/M checkpoint and DNA damage repair upon rereplication

The timely assembly of prereplicative complexes at replication origins is tightly controlled to ensure that genomic DNA is replicated once per cell cycle. The loss of geminin, a DNA replication inhibitor, causes rereplication that activates a G2/M checkpoint in human cancer cells. Fanconi anemia (FA) is an autosomal recessive and X-linked disorder associated with cancer susceptibility. Here we show that rereplication activates the FA pathway both for the activation of a G2/M checkpoint and for repair processes, like recruitment of RAD51. Both ATR and BRCA1 are required to activate the FA pathway. The G2/M checkpoint-mediated arrest of the cell cycle is critical for the prevention of both apoptosis and the accumulation of cells with rereplicated DNA, because the loss of ATR, BRCA1, or FANCA promotes apoptosis and suppresses the accumulation. The accumulation of cells with rereplicated DNA is restored by the artificial induction of a G2-phase arrest even when ATR, BRCA1, or FANCA is absent. Therefore, the ATR- and BRCA1-mediated FA pathway is required for the activation of a G2/M checkpoint and for DNA damage repair in response to the endogenous signal of rereplication. In its absence, the cells rapidly lose viability when faced with rereplication.     

FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway

The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100-deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA-BRCA DNA damage response network.     

FANCL supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.     

Human alpha spectrin II and the Fanconi anemia proteins FANCA and FANCC interact to form a nuclear complex.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital abnormalities, cancer susceptibility, and a marked cellular hypersensitivity to DNA interstrand cross-linking agents, which correlates with a defect in ability to repair this type of damage. We have previously identified an approximately 230-kDa protein present in a nuclear protein complex in normal human lymphoblastoid cells that is involved in repair of DNA interstrand cross-links and shows reduced levels in FA-A cell nuclei. The FANCA gene appears to play a role in the stability or expression of this protein. We now show that p230 is a well known structural protein, human alpha spectrin II (alphaSpIISigma*), and that levels of alphaSpIISigma* are not only significantly reduced in FA-A cells but also in FA-B, FA-C and FA-D cells (i.e. in all FA cell lines tested), suggesting a role for these FA proteins in the stability or expression of alphaSpIISigma*. These studies also show that alphaSpIISigma* forms a complex in the nucleus with the FANCA and FANCC proteins. alphaSpIISigma* may thus act as a scaffold to align or enhance interactions between FA proteins and proteins involved in DNA repair. These results suggest that FA represents a disorder in which there is a deficiency in alphaSpIISigma*.     

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.     

Functional interaction of monoubiquitinated FANCD2 and BRCA2/FANCD1 in chromatin

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2(-/-) cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2(-/-) cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.     

Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased approximately 9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.     

Cross-links between Fanconi anaemia and BRCA2

Fanconi anaemia (FA) is a rare cancer-prone syndrome associated with a defect in the repair of DNA cross-links. Six genes involved in FA have been previously cloned and characterised. Now the two remaining subtypes (FA-B and FA-D1) have been shown by Howlett et al. to be associated with mutations in BRCA2 and to express truncated BRCA2 proteins. Their results suggest that the six cloned FA genes are linked with BRCA2 in a common pathway. Here Steve West discusses some of the implications of these findings.     

Multiple TPR motifs characterize the Fanconi anemia FANCG protein

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.     

Defective cell proliferation is an attribute of overexpressed Notch1 receptor and impaired autophagy in Fanconi Anemia

Fanconi Anemia (FA) is an inherited bone marrow failure syndrome caused by mutation in FA pathway proteins, involved in Interstrand Cross Link (ICL) repair. FA cells exhibit in vitro proliferation arrest due to accumulated DNA damage, hence understanding the rescue mechanism that renders proliferation advantage is required. Gene expression profiling performed in FA patients Peripheral Blood Mononuclear Cells (PBMCs) revealed a wide array of dysregulated biological processes. Functional enrichment and gene clustering analysis showed crippled autophagy process and escalated Notch signalling pathway in FA clinical samples and cell lines. Notch pathway mediators overexpression were reverted in FANCA mutant cells when treated with Rapamycin, an autophagy inducer. Additionally, Rapamycin stabilized cell viability after treatment with the DNA damaging agent, MitomycinC (MMC) and enhanced cell proliferation genes expression in FANCA mutant cells. Inherently FANCA mutant cells express impaired autophagy; thus activation of autophagy channelizes Notch signalling cascade and sustains cell viability.     

Hsp90 and the Fanconi Anemia Pathway: A Molecular Link Between Protein Quality Control and the DNA Damage Response

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an essential role in cell growth and survival. The chaperone exerts these functions by regulating key signaling proteins involved in cell growth/survival and protecting cells from proteotoxic stress. Importantly, Hsp90 inhibitors including geldanamycin analogues show anti-tumor effects. We recently found that Hsp90 promotes stabilization and nuclear localization of the Fanconi anemia (FA) protein FANCA, which is required for activation of the FA pathway. The FA pathway is a multiprotein biochemical pathway involved in genotoxic signaling, defects in which cause genomic instability, hematopoietic stem cell failure and tumor development. Inhibition of Hsp90 impairs the intracellular homeostasis of FANCA, resulting in disruption of the FA pathway. These findings have important implications for rational cancer chemotherapy using Hsp90 inhibitors. We also discuss the possible functions of Hsp90 in FA pathophysiology and stem cell/cancer biology. Based on our findings and other data, we propose that Hsp90 functions as “a guardian of the genome” through the control of DNA repair proteins.     

Resistance to mitomycin C requires direct interaction between the Fanconi anemia proteins FANCA and FANCG in the nucleus through an arginine-rich domain

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, birth defects, and chromosomal instability. Because FA cells are sensitive to mitomycin C (MMC), FA gene products could be involved in cellular defense mechanisms. The FANCA and FANCG proteins deficient in FA groups A and G interact directly with each other. We have localized the mutual interaction domains of these proteins to amino acids 18-29 of FANCA and to two noncontiguous carboxyl-terminal domains of FANCG encompassing amino acids 400-475 and 585-622. Site-directed mutagenesis of FANCA residues 18-29 revealed a novel arginine-rich interaction domain (RRRAWAELLAG). By alanine mutagenesis, Arg(1), Arg(2), and Leu(8) but not Arg(3), Trp(5), and Glu(7) appeared to be critical for binding to FANCG. Similar immunolocalization for FANCA and FANCG suggested that these proteins interact in vivo. Moreover, targeting of FANCA to the nucleus or the cytoplasm with nuclear localization and nuclear export signals, respectively, showed concordance between the localization patterns of FANCA and FANCG. The complementation function of FANCA was abolished by mutations in its FANCG-binding domain. Conversely, stable expression of FANCA mutants encoding intact FANCG interaction domains induced hypersensitivity to MMC in HeLa cells. These results demonstrate that FANCA-FANCG complexes are required for cellular resistance to MMC. Because the FANCC protein deficient in FA group C works within the cytoplasm, we suggest that FANCC and the FANCA-FANCG complexes suppress MMC cytotoxicity within distinct cellular compartments.     

Molecular spectra of HPRT deletion mutations in circulating T-lymphocytes in Fanconi anemia patients

The principal cellular feature of Fanconi anemia (FA), an inherited cancer prone disorder, is a high level of chromosomal breakage, amplified after treatment with crosslinking agents. Three of the eight genes involved in FA have been cloned: FANCA, FANCC and FANCG. However, their biological functions remain unknown. We previously observed an excessive production of deletions at the HPRT locus in FA lymphoblasts belonging to the relatively rare complementation group D(1) and an increased frequency of glycophorin A (GPA) variants in erythrocytes derived from FA patients (2). In thi study, we examined the molecular nature of 31 HPRT mutations formed in vivo in circulating T-lymphocytes isolated from 9 FA male patients. The results show that in all FA patients investigated the deletions are by far the most prevalent mutational event in contrast to age matched healthy donors, in which point mutations predominate. The complementation group in the FA patients examined in the present study has not yet been defined. However, knowing that mutations in the FANCA and FANCC gene are found to be involved in at least 70% of the FA patients, it can be expected that the excessive production of deletions is a general feature of the FA phenotype. In addition, the spectrum of HPRT deletions observed in FA patients differs from that of healthy children: there is a high frequency of 3'-terminal deletions and a strikingly low proportion of V(D)J mediated events. Based on previous findings, a decreased fidelity of coding V(D)J joint formation (3) and an inaccurate repair of specific DNA double strand breaks via Non-Homologous End Joining (4), we propose that FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion pronenness.