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*.     

Human alpha spectrin II and the FANCA, FANCC, and FANCG proteins bind to DNA containing psoralen interstrand cross-links

Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process.     

Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.     

Fanconi Anemia Proteins and the S Phase Checkpoint

DNA interstrand crosslinks (ICLs) repair represents a formidable task for mammalian cells. Indeed, such DNA lesions, bridging both opposite DNA helices, function as a roadblock for every DNA transaction, in particular DNA replication. The eight Fanconi anemia (FA) proteins interact in a common pathway that is thought to be central in ICLs sensing/repair. Interestingly, FA cells, either mutated in one of the proteins composing the FA core complex or in the downstream FA protein FANCD2, exhibited a partial intra-S checkpoint defect in response to crosslinked DNA. Most importantly, the FA proteins work in the ATR-NBS1 branch of the ICL-induced checkpoint pathway as demonstrated by knocking-down CHK1 or MRE11 expression in a FA background. Even though our data disclose a clear functional role for the FA proteins in the intra-S checkpoint response it does not give a definite answer on what FA proteins do in this process and how they participate in the suppression/restart of DNA synthesis.It seems conceivable that FA proteins participate in the process involved in the recovery of stalled replication forks, a common event in proliferating cells, possibly ensuring correct replication fork repair by homologous recombination.     

Chromosomal instability of fanconi anemia cells is not the consequence of a defective repair activity of the ribosomal protein S3.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder characterized by developmental defects, hypersensitivity toward oxygen and DNA crosslinking agents, and susceptibility to cancer. An increased level of reactive oxygen intermediates and an increased level of 8-oxoguanine in FA cells point to a defective oxygen metabolism. Recent investigations showed that FA cells from several complementation groups have a reduced capacity to repair oxidatively damaged DNA. One major enzyme involved in the repair of oxidative DNA lesions is the ribosomal protein S3. Previous reports implied a role for the ribosomal protein S3 in DNA repair in FA cells. However, a more detailed analysis of the ribosomal protein S3 in FA cells from complementation groups A-E could not confirm this. DNA analysis and Western blot analysis did not show significant differences in ribosomal protein S3 between FA cells and cells from healthy individuals. Furthermore, even the overexpression of the ribosomal protein S3 did not reduce the chromosomal instability of FA cells.     

Regulatory functions of ubiquitin in diverse DNA damage responses

In recent years there has been intense investigation and rapid progress in our understanding of the cellular responses to various types of endogenous and exogenous DNA damage that ensure genetic stability. These studies have identified numerous roles for ubiquitylation, the post-translational modification of proteins with single ubiquitin or poly-ubiquitin chains. Initially discovered for its role in targeting proteins for degradation in the proteasome, ubiquitylation functions in a variety of regulatory roles to co-ordinate the recruitment and activity of a large number of protein complexes required for recovery from DNA damage. This includes the identification of essential DNA damage response genes that encode proteins directly involved in the ubiquitylation process itself, proteins that are targets for ubiquitylation, proteins that contain ubiquitin binding domains, as well as proteins involved in the de-ubiquitylation process. This review will focus on the regulatory functions of ubiquitylation in three distinct DNA damage responses that involve ubiquitin modification of proliferating cell nuclear antigen (PCNA) in DNA damage tolerance, the core histone H2A and its variant H2AX in double strand break repair (DSBR) and the Fanconi anaemia (FA) proteins FANCD2 and FANCI in cross link repair.     

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.     

The DNA topoisomerase IIbeta binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.     

The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair

The Fanconi anemia (FA) protein FANCC is essential for chromosome stability in vertebrate cells, a feature underscored by the extreme sensitivity of FANCC-deficient cells to agents that crosslink DNA. However, it is not known how this FA protein facilitates the repair of both endogenously acquired and mutagen-induced DNA damage. Here, we use the model vertebrate cell line DT40 to address this question. We discover that apart from functioning in homologous recombination, FANCC also promotes the mutational repair of endogenously generated abasic sites. Moreover in these vertebrate cells, the efficient repair of crosslinks requires the combined functions of FANCC, translesion synthesis, and homologous recombination. These studies reveal that the FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA.     

Insights into Fanconi Anaemia from the structure of human FANCE

Fanconi Anaemia (FA) is a cancer predisposition disorder characterized by spontaneous chromosome breakage and high cellular sensitivity to genotoxic agents. In response to DNA damage, a multi-subunit assembly of FA proteins, the FA core complex, monoubiquitinates the downstream FANCD2 protein. The FANCE protein plays an essential role in the FA process of DNA repair as the FANCD2-binding component of the FA core complex. Here we report a crystallographic and biological study of human FANCE. The first structure of a FA protein reveals the presence of a repeated helical motif that provides a template for the structural rationalization of other proteins defective in Fanconi Anaemia. The portion of FANCE defined by our crystallographic analysis is sufficient for interaction with FANCD2, yielding structural information into the mode of FANCD2 recruitment to the FA core complex. Disease-associated mutations disrupt the FANCE–FANCD2 interaction, providing structural insight into the molecular mechanisms of FA pathogenesis.     

Changes in protein expression due to deleterious mutations in the FA/BRCA pathway

Inherited deleterious mutations in one of the Fanconi anemia genes lead to a disease, characterized by bone marrow failure, myeloid leukemia, and hypersensitivity to DNA damage. We identified proteins likely associated to the molecular signaling pathways involved in DNA repair of interstrand cross-link lesions and in mechanisms of genomic stability mediated by FA/BRCA pathways. We compared protein maps resolved by bidimensional electrophoresis and analyzed differentially expressed proteins, by mass spectrometry, between FA complementation group C (FANCC)-deficient cells, and their ectopically corrected counterpart in physiological conditions or after treatment with MMC. We found six differentially expressed proteins; among them, the checkpoint mediator protein MDC1 whose expression was disrupted in FANCC^−/− cells. The potential role of differentially expressed proteins in FA phenotype is discussed.     

DNA repair: exploiting the Fanconi anemia pathway as a potential therapeutic target

DNA repair is an active cellular process to respond to constant DNA damage caused by metabolic processes and environmental factors. Since the outcome of DNA damage is generally adverse and long term effects may contribute to oncogenesis, cells have developed a variety of DNA repair mechanisms, which operate depending on the type of DNA damage inflicted. At least 15 Fanconi anemia (FA) proteins interact in a common pathway involved in homologous recombination. Inherited homozygous mutations in any of these FA genes cause a rare disease, Fanconi anemia, characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility. Heterozygous germline FA mutations predispose to various types of cancer. In addition, somatic FA mutations have been identified in diverse cancer types. Evidence exists that cells deficient in the FA pathway become dependent on alternative pathways for survival. Additional inhibition of such alternative pathways is thus expected to result in cell death, creating a relationship of synthetic lethality. Identifying these relationships can reveal yet unknown mechanisms of DNA repair and new targets for therapy.     

The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway

Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2.     

The Fanconi anemia pathway limits the severity of mutagenesis

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.     

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.     

Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage

The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.     

Deficiency in incisions produced by XPF at the site of a DNA interstrand cross-link in Fanconi anemia cells

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.     

What is wrong with Fanconi anemia cells?

Figuring out what is wrong in Fanconi anemia (FA) patient cells is critical to understanding the contributions of the FA pathway to DNA repair and tumor suppression. Although FA patients exhibit a wide range of disease manifestation as well as severity (asymptomatic to congenital abnormalities, bone marrow failure, and cancer), cells from FA patients share underlying defects in their ability to process DNA lesions that interfere with DNA replication. In particular, FA cells are very sensitive to agents that induce DNA interstrand crosslinks (ICLs). The cause of this pronounced ICL sensitivity is not fully understood, but has been linked to the aberrant activation of DNA damage repair proteins, checkpoints and pathways. Thus, regulation of these responses through coordination of repair processing at stalled replication forks is an essential function of the FA pathway. Here, we briefly summarize some of the aberrant DNA damage responses contributing to defects in FA cells, and detail the newly-identified relationship between FA and the mismatch repair protein, MSH2. Understanding the contribution of MSH2 and/or other proteins to the replication problem in FA cells will be key to assessing therapeutic options to improve the health of FA patients. Moreover, loss of these factors, if linked to improved replication, could be a key event in the progression of FA cells to cancer cells. Likewise, loss of these factors could synergize to enhance tumorigenesis or confer chemoresistance in tumors defective in FA-BRCA pathway proteins and provide a basis for biomarkers for disease progression and response.     

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.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.