Studies on cancer-prone and rare human genetic disorders often lead to significant advances in our understanding of the complex network of genome stability and DNA repair pathways that have evolved in the human genome to prevent the harmful effects of exposure to DNA damaging agents. One such disorder is Fanconi Anemia, an autosomal recessive disease characterized by an increased spontaneous and DNA cross-linkers induced chromosome instability, progressive pancytopenia and cancer susceptibility. At least eleven genes are involved in Fanconi anemia, including the breast cancer susceptibility gene BRCA2. Six of the Fanconi anemia proteins (FANCA, C, E, F, G and L) assemble in a complex that is required for FANCD2 activation by monoubiquitination in response to DNA damage or during S-phase progression. Active FANCD2 then colocalizes with the product of the breast cancer susceptibility gene BRCA1 in discrete nuclear foci. FANCD2 is also independently phosphorylated by ATM in response to ionising radiation and interacts with the MRE11/Rad50/NBS1 complex, which is directly involved in homologous recombination DNA repair pathway and in cell cycle checkpoint response to DNA damage. Available data indicate that FANCD2 is involved in cell cycle regulation and DNA repair. Our current knowledge on the functional significance of FA pathway and more specifically FANCD2 and its interacting proteins in pathways of genomic surveillance and maintenance will be discussed in this chapter.

Introduction

In 1967 a Swiss physician, Guido Fanconi, reported his observations of a recessively inherited aplastic anemia in two brothers along with several other physical malformations.¹ Fanconi anemia (FA) is a rare syndrome characterized by progressive pancytopenia, increased spontaneous and mutagen-induced chromosome instability, congenital malformations and cancer susceptibility. The phenotype of the FA patients reflects that the genes involved in FA are important for chromosome stability, normal embryo development and for the preservation of several types of stem cells. The most frequent birth malformations in FA are growing retardation and abnormalities in the skin (typically cafe-au-lait spots), upper extremities (specially in the thumbs and forearms), kidneys and gastrointestinal system.² As 30% of the patients have no signs of physical flaws,³ the ability of DNA cross-linking chemicals such as mitomycin C (MMC) and diepoxybutane (DEB) to induce chromosome aberrations in the patients' blood lymphocytes² must be tested for the final confirmation of FA phenotype. The confirmation of FA diagnosis requires cell culturing of peripheral blood lymphocytes, MMC or DEB treatment, analysis of the metaphases and detection of chromosome aberrations, usually of chromatid type such as radial figures (fig. 1).

Figure 1

Typical chromatid-type chromosomal aberrations found in FA cells.

FA is a very heterogeneous disease with at least eleven complementation groups described until now⁴ (see below). The assignment of a FA patient to a given complementation group is classically resolved by cell fusion-based complementation studies (fig. 2). The cloning of the genes involved in the most prevalent complementation groups has more recently permitted to develop techniques of genetic subtyping by retroviral transduction of wild type FA genes. Primary blood lymphocytes or cell lines retrovirally transduced with the cDNAs of the various FA genes are tested for the phenotypic correction of MMC hypersensitivity.⁵

Figure 2

Complementation group determination in FA by cell fusion. A) When cells derived from a patient of an unknown complementation group (yellow) are fused with cells of a known but different complementation group (blue) the FA phenotype is functionally complemented (more...)

FA is usually a fatal disease with a mean survival of twenty-four years.⁶ Over 90% of FA patients will most probably develop hematological complications (bone marrow failure, aplastic anemia, thrombocytopenia or pancytopenia) at pediatric age.⁶ The cumulative incidence by 40 years of age is 33% for hematologic malignancies and 28% for solid tumors, with a strong predisposition to squamous cells carcinoma, especially of the head and neck region. In fact, there is a 500-fold increase in the cumulative incidence of head and neck cancer in FA patients compared with the normal population.⁶

The phenotype of primary or immortalized FA-derived cell lines is characterized by their extreme sensitivity to agents that produce DNA interstrand crosslinks such as MMC, DEB and cisplatin, resembling the phenotype of the patients' blood lymphocytes. After treatment with these agents, FA cell lines show increased chromosome instability. Another feature observed in FA cell lines is a delay in G2/M or late S-phase of the cell cycle. This delay occurs spontaneously and increases greatly after treatment of the cells with crosslinking agents.⁷

The Genetics of Fanconi Anemia

FA is a very rare autosomal recessive genetic disease with a prevalence of 1-5 per million.² The frequency of heterozygote carriers in the population is estimated to vary between 1:200 to 1:300. Some ethnic groups have a higher prevalence of FA due to a founder effect and consanguinity. Two examples are the Afrikaner population of South Africa and the Ashkenazi Jewish with a carrier frequency of 1/77 and 1/90, respectively. An even higher prevalence of FA is found in Spanish Gypsies (Callen et al, manuscript submitted). It is known that a common mutation in the FANCC gene is responsible for the majority of FA patients in Ashkenazi Jews⁸ and similarly the majority of the patients of the Afrikaner population of South Africa have an identical mutation in FANCA. Molecular and genealogical evidences obtained in this later group confirmed the existence of a founder effect for FA in South Africa.⁹

Cell fusion complementation studies (fig. 2) revealed the existence of at least eleven FA complementation groups: FANCA, FANCB, FANCC, BRCA2/FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCL, FANCI and FANCJ. Most of the FA genes, with the exception of FANCB, FANCI and FANCJ, have been cloned and characterized.^4,10,11 According to the International FA Register, complementation groups A (65%), C (15%), and G (10%) account for at least 90% of the FA patients in a population of 755 FA patients from North America while the rest of the subtypes are relatively rare.^4,6 Cell lines derived from FANCD1 patients have biallelic mutations in the breast cancer susceptibility gene BRCA2 and express truncated BRCA2 proteins.¹² The BRCA2 protein participates in DNA repair by homologous recombination. ^13-15 Mutations in a DNA repair gene, BRCA2, in cells derived from FANCD1 patients strongly suggest that at least a subset of FA phenotype is causally linked to DNA repair deficiency. Recently, afflicted patients in four FA families were found to be compound heterozygotes for BRCA2 mutations. The individuals affected, exhibit the classical FA phenotype (higher levels of spontaneous and chemically induced chromosome aberrations and hematological complications) together with brain tumour development. In one of the kindreds, a family history for breast cancer was also observed. The cooccurrence of FA phenotype, brain tumours and breast cancer constitutes a new syndromic association and highlights the critical link between FA pathway and cancer development.¹⁶

FANCD2 As a Key Player in Fanconi Anemia

The molecular biology of FA is steadily becoming clearer and now it is well established that most of the FA proteins (A, C, E, F, G, L and perhaps I) assemble in a nuclear complex required for the activation of FANCD2 via monoubiquitination at lysine 561.^10,11,17 Monoubiquitinated FANCD2 (FANCD2-L) is bigger than its inactive isoform, (FANCD2-S) which can be easily distinguished by western blot analysis. When one of the genes in the FA nuclear complex is mutated, this complex is not properly formed resulting in a single FANCD2-S band in a western blot (fig. 3). An exception to this is FANCI, which although dispensable for FA core complex formation as judged by coimmunoprecipitation studies, is required for FANCD2 monoubiquitination indicating that FANCI is upstream of FANCD2. In contrast, FANCJ and FANCD1/BRCA2 deficient cells are able to form a FA multiprotein core complex and to monoubiquitinate FANCD2, suggesting that their defect in the FA pathway is downstream to the crucial step of FANCD2 activation.⁴ The importance of FANCD2 as one of the key players of FA pathway is illustrated by the evolutionary conservation of FANCD2 together with FANCL¹¹ in distant species such as Drosophila melanogaster, Caenorabditis elegans, Arabidopsis thaliana.¹⁸ Cloning and sequencing of the Drosophila FANCD2 gene, for instance, revealed a remarkable functional conservation of important features, such as the residue K561, during evolution¹⁹ (fig. 4).

Figure 3

FA nuclear complex and activation of FANCD2. The FA nuclear complex mediates the activation by monoubiquitination of FANCD2 and western blot with antibodies against FANCD2 reveals two bands (FANCD2-L and S) in normal cells (lane 1), one FANCD2-S (short) (more...)

Figure 4

Human and Drosophila FANCD2 post-translational modification sites. Sequence and structural biocomputational analysis indicated a 23% of identity and 43% of similarity between human (HD2) and the Drosophila (DD2) FANCD2 proteins. Human ATM phosphorylation (more...)

The monoubiquitinated isoform of FANCD2 associates with the repair protein BRCA1 in DNA damage-induced nuclear foci.¹⁷ This foci formation is induced not only by crosslinking agents but also by other DNA damaging agents such as UVC light and ionising radiation.¹⁷ This evidence suggests a wider field of action for FANCD2 probably in cooperation with other DNA damage response proteins known to also colocalize in nuclear foci with BRCA1 after DNA damage.²⁰ In addition, FANCD2 undergoes monoubiquitination during the S-phase of the cell cycle even in the absence of mutagen induced-DNA damage and the monoubiquitinated FANCD2 colocalizes with BRCA1 and RAD51 in S-phase-specific nuclear foci.²¹

Recent studies have shown that ubiquitin ligase activity of BRCA1²² is not directly responsible of FANCD2 monoubiquitination, since this post-translational modification also occurs in BRCA1 deficient cells.²³ The main catalytic subunit involved in FANCD2 monoubiquitination is the gene involved in the complementation group FA-L (FANCL) known as ubiquitin ligase PHF9. FANCL-PHF9 possesses E3 ubiquitin ligase activity in vitro and it has a crucial role in the FA pathway.¹¹

In addition to monoubiquitination, a second posttranslational modification of FANCD2 also seems to be critical for DNA damage response. ATM, the product of the gene mutated in the chromosome fragility syndrome ataxia telangiectasia, phosphorylates FANCD2 at serine 222 in response to ionising radiation and activates G1-S checkpoint control.²⁴ Ser 222 is also highly conserved in evolution.¹⁹ The ATM gene encodes for a protein kinase triggered by ionising radiation that phosphorylates a number of downstream targets such as the tumour suppressor gene p53, the Nijmegen breakage syndrome (NBS) protein NBS1, CHK2 and BRCA1, all of which are involved in the S-phase checkpoint activated by ionising radiation. Biallelic loss of ATM gene or of one of its substrates produces a defect in this S-phase checkpoint characterized by a radioresistant DNA synthesis after exposure to ionising radiation.²⁵ Among the FA cell lines, only FANCD2 are mildly sensitive to ionising radiation and are defective in the S-phase checkpoint induced by ionising radiation.²⁴ Thus, it appears that the two post-translational modifications of FANCD2 are important for DNA repair and intra-S-phase checkpoint regulation after DNA damage. The two posttranslational modifications are independent: disruption of the FA complex results in the lack of the activation of FANCD2 by monoubiquitination and in hypersensitivity to cross-linking agents, without affecting the S-phase checkpoint. On the other hand, the lack of ATM-dependent FANCD2 phosphorylation leads to an inactivation of an S-phase cell cycle checkpoint and to radioresistant DNA synthesis, but does not affect FANCD2 monoubiquitination by the FA complex after cross-linkers induced DNA damage. Further supporting this hypothesis is the fact that ATM cell lines are not hypersensitive to cross-linkers and all the FA cells with the only exception of FANCD2, are not hypersensitive to ionising radiations. These observation suggest that FANCD2 could be where the FA and ATM signaling pathways converge.²⁴ ATM-dependent response to ionising radiations is not only mediated through the interaction with FANCD2 but also by NBS1 which is phosphorylated by ATM. After phosphorylation, NBS1 interacts with FANCD2 promoting its ATM-mediated phosphorylation and activating the S-phase cell cycle checkpoint and radioresistant DNA synthesis. Accordingly, ATM-dependent phosphorylation of FANCD2 is defective in NBS1 deficient cells (fig. 6B).²⁶

Figure 6

FANCD2 is a central node between repair and cell cycle response to DNA damage. A) In response to DNA damage induced by DNA crosslinkers, the FA nuclear complex activates FANCD2 monoubiquitination and, via FANCC, the RMN complex assembly. FANCD2, the RMN (more...)

High FANCD2 expression was found in human germ cells and in haematopoietic system and other highly proliferative tissues known to be predisposed to cancer development in FA patients, particularly in the head and neck region and in the uterine cervix. These observations point to an important role for FANCD2 in the maintenance of genomic integrity during cellular proliferation within these tissues.²⁷

DNA Repair and Cell Cycle Regulation Downstream FANCD2

The phenotype of the FA patients tells us how the FA pathway is important in genome stability, embryonic development and in the maintenance of several types of stem cells. Cultured cells from FA patients display a high level of spontaneous chromosome breaks and an increased frequency of intragenic deletions, suggesting that FA cells may have deficiencies in the repair of DNA double strand breaks in addition to DNA cross links. Eukaryotic cells possess two modes of repair pathways for dealing with double strand breaks: (1) nonhomologous end-joining pathway (NHEJ) and (2) homologous recombination (HR) repair. Among these two, NHEJ is relatively more simple where the DNA bearing a double strand break is simply ligated back.²⁸ It has been observed that the FA cell lines are impaired in the DNA repair by NHEJ as they show abnormal rearrangements associated with VDJ recombination²⁹ and impaired fidelity in blunt DNA end joining.^30,31 A deficient DNA end joining activity in extracts from FA fibroblasts has also been reported. The Fanconi anemia extracts had 3- to 9-fold less DNA end joining activity and rejoined substrates with significantly less fidelity than normal extracts. Protein expression levels of the DNA-dependent protein kinase (DNA-PK)/Ku-dependent NHEJ proteins Xrcc4, DNA ligase IV, Ku70, and Ku86 in FA and normal extracts were indistinguishable. Taken together, these results suggest that the FA fibroblast extracts have a deficiency in a DNA end joining process that is distinct from the DNA-PK/Ku-dependent NHEJ pathway.³²

The HR repair is an error-free DNA repair system specific of S and G2-phases of the cell cycle, when two chromatids are present. In this repair process, the undamaged chromatid is used as a template for the error free exchange and repair of the other chromatid (fig. 5). As mentioned earlier, cell lines derived from FANCD1 patients have biallelic mutations in BRCA2, and FANCD1 cells express truncated BRCA2 proteins.¹² This finding is particularly relevant from the DNA repair point of view because several studies have implicated BRCA2 in HR repair during S-phase.^13-15 These observations convincingly demonstrate that a potential DNA repair gene, BRCA2, is also implicated in the pathogenesis of FA. Research on the roles of BRCA1 and BRCA2 led to the finding that both proteins interact with the recombinase Rad51 and all of them are involved in HR pathway.³³ The findings that BRCA2/FANCD1 and Rad51 are downstream in the FA/BRCA pathway and that FANCD2 forms nuclear foci with BRCA1 and Rad51 during S-phase, raise the possibility that the FA/BRCA pathway play a role in HR repair of interstrand crosslinks and double strand breaks during S-phase.^17,21 Cells impaired in HR repair such as BRCA1 and BRCA2 deficient cells are very sensitive to DNA crosslinking agents indicating that HR is important in the processing of DNA crosslinks.³⁴ Functional linkage between the FA pathway and the breast cancer susceptibility genes has been demonstrated by the interaction of FANCD2 with BRCA1¹⁷ and by the discovery that the FANCD1 gene is identical to BRCA2.¹² Also other FA proteins interact with BRCA genes: an interaction between FANCA and BRCA1³⁵ and binding of FANCG to two separate sites of BRCA2, located on either side of the BRC repeats have been demonstrated using yeast two-hybrid system. Furthermore, FANCG can be coimmunoprecipitated with BRCA2 from human cell extracts and FANCG colocalizes in the nuclear foci with both BRCA2 and RAD51 following MMC-induced DNA damage.³⁶ A role for FA proteins in DNA repair is further strengthened by their association with BLM helicase whose unwinding activity is critical for DNA repair activities. A complex isolated from HeLa cell extract using antibody to Bloom syndrome protein (BLM) has been shown to contain at least five of the FA complementation group proteins (A, C, G, E and F). This complex exhibits a DNA-unwinding activity, predominantly owing to the presence of BLM helicase because such a complex isolated from BLM-deficient cells lack such an activity. These results have been confirmed by the fact that the same complex could be isolated with an antibody against FANCA. It is interesting to note that cells from Bloom syndrome patients are characterized by spontaneous and UV induced elevation of sister chromatid exchanges which are attributed to a deficiency in HR pathway. Coimmunoprecipitation of FA proteins with BLM in the same complex suggests that both BLM and FA proteins may operate in the same repair pathway.³⁷ As previously mentioned, ATM directly phosphorylates FANCD2 on Ser 222 which is required for the activation of S-phase checkpoint.²⁴ ATM not only phosphorylates FANCD2 but also an increasing number of proteins involved in both HR and NHEJ.³⁸ NBS1 is phosphorylated by ATM on Ser 343 and forms a complex with MRE11 and Rad50 (RMN complex). The RMN complex is a very important regulator of cell cycle checkpoint and DNA repair responses in all eukaryotic cells and the complex is directly involved in HR.^39-41 A subset of NBS patients shares symptoms with FA patients such as bone marrow failure⁴² and some NBS cell lines shows a mild sensitivity to MMC.²⁶ The identification of a patient with an atypical FA phenotype bearing biallelic mutations in NBS1 gene conclusively pointed out an interaction between FA and NBS.²⁶ In support, NBS1 and the monoubiquitinated isoform of FANCD2 colocalize in sub-nuclear foci in response to DNA damage by crosslinking agents. The disruption of the monoubiquitination site on FANCD2 or the disruption of the MRE11-interacting carboxyl terminus of NBS1 prevents these foci to form, resulting in MMC hypersensitivity.²⁶ Hence the FA pathway and the RMN complex must cooperate in the cellular response to DNA damage by crosslinking agents (fig.6A). After ionising radiation treatment, NBS1 is phosphorylated by ATM and the phosphorylated form of NBS1 promotes the subsequent phosphorylation of FANCD2 by ATM, since ATM-dependent phosphorylation of FANCD2 is defective in Nbs1 cells (fig.6B) Therefore, FANCD2 and the RMN complex also cooperate in the checkpoint response after DNA damage by ionising radiation. All these observations indicate that the FA pathway in general and FANCD2 in particular function at the intersection of two inter-dependent DNA repair and cell cycle signaling pathways initiated by two DNA damaging agents. In response to MMC and other crosslinking agents, active FANCD2 assembles with BRCA1, MRE11 complex, BRCA2 and Rad51 in nuclear foci during HR (fig. 6A). In case of radiation treatment, ATM phosphorylates FANCD2 and NBS1 thereby imposing the intra-S-phase checkpoint regulation²⁶ (fig. 6B). The interaction with the RMN complex is not only a prerogative for FANCD2, since RMN assembly is defective in FANCC cells in response to MMC. These alterations in the assembly of DNA-repair proteins and cell cycle regulators in FA can explain the DNA-damage processing anomalies observed in FA cells and for the genetic instability and the cancer predisposition of this syndrome⁴³ (Fig. 6A).

Figure 5

Schematic representation of homologous-recombination repair during S-phase. DNA damage activates ATM/ATR that triggers by phosphorylation the RAD50/MRE11/NBS1 complex. The 5'—3' exonuclease activity of this complex exposes both 3' ends (A). To (more...)

Concluding Remarks

The main conclusion that arises from this chapter is that the FA pathway, and especially FANCD2, is a central player in a network of DNA repair and checkpoint responses to DNA damage, but several specific questions remain unanswered. What is the exact molecular nature of the relationship between the FA pathway and HR repair? There are numerous indications and evidences of this interaction but what exactly the FA pathway and FANCD2 do remains unknown. What is the role of FA pathway in the other phases of the cell cycle other than S-phase? In the last years, the majority of studies have focused on the role of the FA pathway in S-phase and in HR, but we must not forget that this is only the tip of the iceberg. MMC exerts its DNA damaging action only in S-phase, but the FA pathway is activated by a wide array of chemical and physical agents—such as UVC and ionising radiation for instance¹⁷—that produce damage repaired by mechanisms (nucleotide excision repair, base excision repair, NHEJ) that operate not only in S-phase but also in G1 and G2 phases. Additionally, we must bear in mind that a defect in any of the FA proteins has a profound impact on so many fundamental cellular activities that trying to explain all of these outcomes only on the basis of a deficiency in FANCD2 activation, is probably too simplistic, if not misleading at all. There are an amazing number of studies focused on the many additional roles that the other FA proteins undertake both in the cytoplasm and in the nucleus, such as the processing of reactive oxygen species, the regulation of apoptosis and the maintenance of telomere integrity.^10,70 Even so the FA/BRCA pathway⁷¹ can be considered as the most exciting discovery of the last few years in the field of FA, and clarifying the many twists and turns of this pathway would prove to be an exciting scientific challenge for the years to come.

Acknowledgements

FA research in our Group is in part supported by the Generalitat de Catalunya (project SGR-00197-2002), the Spanish Ministry of Health and Consumption (projects FIS PI020145 and FIS-Red G03/073), the Spanish Ministry of Science and Technology (projects SAF 2002-03234, SAF2002-11833-E and SAF 2003-00328) and the Commission of the European Union (projects FIGH-CT-2002-00217, FI6R-CT-2003-508842 and HPMF-CT-2001-01330). M.B. is supported by a long-term postdoctoral Marie Curie fellowship awarded by the Commission of the European Union. J.S. is supported by a “Ramön y Cajal” project entitled “Genome stability and DNA repair” cofinanced by the Spanish Ministry of Science and Technology and the Universitat Autònoma de Barcelona.

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