Fanconi anemia and DNA repair - Semantic Scholar

© 2001 Oxford University Press

Human Molecular Genetics, 2001, Vol. 10, No. 20 2253–2259

Fanconi anemia and DNA repair Markus Grompe* and Alan D’Andrea1 Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road L103, Portland, OR 97201, USA and 1Division of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA Received July 10, 2001; Accepted July 24, 2001

Fanconi anemia (FA) is an autosomal recessive disorder caused by defects in at least eight distinct genes FANCA, B, C, D1, D2, E, F and G. The clinical phenotype of all FA complementation groups is similar and is characterized by progressive bone marrow failure, cancer proneness and typical birth defects. The principal cellular phenotype is hypersensitivity to DNA damage, particularly interstrand DNA crosslinks. The FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. Five of the FA proteins (FANCA, C, E, F and G) interact in a nuclear complex upstream of FANCD2. FANCB and FANCD1 have not yet been cloned, but it is likely that FANCB is part of the nuclear complex and that FANCD1 acts downstream of FANCD2. The FA nuclear complex regulates the mono-ubiquitination of FANCD2 in response to DNA damage, resulting in targeting of this protein into nuclear foci. These foci also contain BRCA1 and other DNA damage response proteins. In male meiosis, FANCD2 also co-localizes with BRCA1 at synaptonemal complexes. Together, these data suggest that the FA pathway functions primarily as a DNA damage response system, although its exact role (direct involvement in DNA repair versus indirect, facilitating role) has not yet been defined.

INTRODUCTION Fanconi anemia (FA) is an autosomal recessive disorder with a complex organismal and cellular phenotype (1). Although much progress has been made in the understanding of the disorder in recent years, the precise function of the FA proteins remains unknown. In this review, we will discuss the current state of the field and highlight some of the potential functions of the pathway with an emphasis on DNA repair.

Table 1. Frequent birth defects in FA Radial ray defects (absent thumb and/or radius) Short stature Microcephaly Microphthalmia (and other facial dysmorphic features) Urinary tract defects Skin hyperpigmentation/café-au-lait spots Hypogonadism (males)

CLINICAL PHENOTYPES

Gastrointestinal malformations

The clinical hallmark of FA is bone marrow failure, usually starting in childhood (1–3). The anemia is caused by a progressive loss of hematopoietic stem cells and thus affects all blood lineages. Another consistent feature of FA is a high propensity toward malignancy, particularly acute myelogenous leukemia (AML) and squamous cell carcinoma (4,5). A wide variety of birth defects can also occur in FA, but are variable even within the same family (6). The most common defects are listed in Table 1. Together, these clinical manifestations of FA result in a markedly reduced life expectancy with death most frequently due to hematological complications or cancer.

Congenital heart disease

CELLULAR PHENOTYPES Many cellular phenotypes have been reported in FA cells, but the most consistent and accepted of these is hypersensitivity to

Sensoneuronary hearing loss

agents which produce interstrand DNA cross-links (ICL) such as mitomycin C (MMC) or diepoxybutane (DEB) (7). After ICL treatment FA cells display several phenotypes. These include increased chromosome breakage, radial formation and other cytogenetic abnormalities seen in metaphase chromosome spreads. This property is used as the diagnostic test (8,9). In addition, the hypersensitivity can manifest itself as apoptosis or growth arrest depending on cell type. FA cells also have more modest hypersensitivity to other DNA damaging agents such as ionizing radiation (10–12) and oxygen (13–17). A second phenotype, which has been established by many studies, is an increase of the proportion of cells with 4N DNA content (18–24).

*To whom correspondence should be addressed. Tel: +1 503 494 6888; Fax: +1 503 494 6886; Email: [email protected]

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This has generally been interpreted as a G2/M delay. The increase of cells with 4N DNA content can occur spontaneously in some FA cells, but becomes much more pronounced after ICL treatment. Some reports also indicate that FA cells may have primary defects in signaling and apoptosis (25–28) particularly in response to mitoinhibitory cytokines (28–30). The relationship of these other phenotypes to DNA damage is not clear. GENETICS Cell fusion experiments have shown the existence of at least eight separate complementation groups: A, B, C, D1, D2, E, F and G (31–33). To date, six of the corresponding genes have been cloned (33–39). The genes are not clustered but widely dispersed throughout the genome. In most cases, the cloning was achieved by complementation of the MMC sensitivity by cDNA libraries (FANCC, A, G, F and E in chronological order). Two of the genes were isolated by positional cloning approaches (FANCA was cloned independently by this method and FANCD2). Complementation groups A (∼65%), C (∼5–15%) and G (10–15%) account for the majority of patients in most populations (32,40–43). Some ethnic groups have founder mutations accounting for the majority of cases in these populations. The FANCC IVS4+4 a→t allele is prevalent among Ashkenazi Jews with a carrier frequency of ∼1/150 (44–46). Similarly, FANCA is common in the Afrikaners of South Africa due to a founder effect (47). Overall, however, FA is genetically very heterogeneous, with many mutations reported in each of the complementation groups. A mutation database is available on the Internet (http://www.rockefeller.edu/ fanconi/mutate/). There is only a modest correlation between phenotypic differences and the complementation group. Hearing deficits, for example, are found more common in FA-G patients (48). Nonetheless, the complete spectrum of phenotypic variability can be found within single complementation groups and correlates strongly with specific mutations (49). For example, homozygosity for the FANCC IVS4+4 a→t mutation always causes severe FA with a high frequency of birth defects and early onset of the anemia. In contrast, patients homozygous for the FANCC 322∆G mutation have very few birth defects and develop anemia late (49,50). FA PROTEINS All six cloned FA proteins are ‘orphan proteins’ in that their primary amino acid sequence has not revealed any domains of known function. Furthermore, none of the FA proteins has homologues in prokaryotes such as Escherichia coli or single cell eukaryotes such as Saccharomyces cerevisiae. The FANCA, C, G, E and F proteins have no recognized orthologues in nonvertebrates, but the recently isolated FANCD2 protein has a high degree of homology to predicted proteins in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) (33). However, the homologous proteins have no known function in these organisms. Importantly, FANCG was found to be identical to XRCC9, the gene defective in a radiationsensitive Chinese hamster ovary cell line (37).

Figure 1. Interaction of the FA proteins in a common pathway. The FA proteins, FANCA, FANCC, FANCF and FANCG are components of a stable nuclear complex. FANCE interacts with this complex in vitro and is probably associated with the complex in vivo as well. The (uncloned) FANCB protein is also required for the assembly or stabilization of the complex and may be another subunit. Activation of the complex by DNA damage or DNA replication results in the enzymatic modification (mono-ubiquitination at lysine 561) of the FANCD2 protein. The FA protein complex may be a multisubunit mono-ubiquitin ligase itself or may regulate such an activity. The activated FANCD2 protein is subsequently targeted to nuclear foci where it interacts with the BRCA1 protein and other proteins involved in DNA repair. The (uncloned) FANCD1 functions either downstream or independently of the FA pathway.

The FA nuclear complex FANCC protein can be found in both the nucleus and cytoplasm, and it has therefore been somewhat controversial in which compartment it functions (51–54). However, the cloning of other FA genes quickly led to the discovery of interactions between FA proteins in the nucleus. First, FANCA and FANCC were found to interact in a complex (Fig. 1) (55). Later, FANCA, FANCC and FANCG were shown to be associated in a complex (56,57), and the interacting protein domains have been mapped. Most recently, the participation of FANCF (58) and FANCE (59) in the complex was demonstrated. These interactions have also been confirmed in yeast-2 hybrid analysis (59–61). Interestingly, the formation of the FA nuclear complex is disrupted in cells from FA complementation groups A, B, C, E, F and G (56,62). Cells from groups D1 and D2 are the exception, because the nuclear complex is present as in wild-type cells. It should be emphasized that the complex is found only in the nucleus of cells, not the cytoplasm. To date, no signals which lead to the assembly of the FA complex have been found. The complex appears to be constitutively present showing no response to DNA damage or the cell cycle. However, a recent study suggests that the FA nuclear complex is compartmentalized within the nucleus and that a fraction of the complex translocates into the chromatin compartment following cellular exposure to ICL damage (63). Yeast-2-hybrid screens and other techniques have resulted in the identification of several non-FA proteins which potentially interact with FA proteins (25,64–69). Some of these proteins reported to interact with FANCC play a role in cellular redox regulation (25,67). Another FANCC interacting protein, FAZF, is a close homologue of proteins known to function by mediating chromatin remodeling transcriptional modifiers (65).

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FANCD2

Model of the FA pathway: unresolved questions

FANCD2 is not part of the FA nuclear complex and is not required for its assembly or stability. Current evidence suggests that FANCD2 is a target of the function of the FA nuclear complex and acts downstream in the FA pathway (Fig. 1) (70). FANCD2 is exclusively nuclear in location and is present in wild-type cells in two isoforms, FANCD2-L (long) and FANCD2-S (short) with apparent molecular weights of 162 and 155 kDa, respectively (33). The long isoform (FANCD2-L) is mono-ubiquitinated at lysine 561 (70). Importantly, this FANCD2-L is absent in cells from FA complementation groups A, B, C, E, F and G, i.e. all those complementation groups in which the FA nuclear complex is defective (70). This observation suggests that the FA nuclear complex may function as a novel ubiquitin ligase or regulate the activity of such an enzyme. The mono-ubiquitination of FANCD2 is induced by DNA damaging agents, most notably interstrand DNA cross-linkers and ionizing radiation (IR), the same agents to which FA cells are sensitive (70). These treatments lead to an increase of FANCD2 within a short time-frame, only 30 min in the case of IR. Together, these observations suggest that the FA nuclear complex ‘senses’ the presence of DNA damage and then mediates the mono-ubiquitination of FANCD2. The precise biochemical function(s) of FANCD2 is presently unknown, but some deductions can be made from its subcellular localization. FANCD2 immunocytochemistry displays two distinct patterns of staining: diffuse nuclear and nuclear foci. The number and intensity of FANCD2 nuclear foci increases rapidly after DNA damage (70). Importantly, cells from FA groups A, B, C, E, F and G showed only the diffuse nuclear staining pattern and lacked nuclear speckles. Thus, there is a tight correlation between mono-ubiquitination of FANCD2 and the presence of nuclear foci. Mono-ubiquitination serves as targeting signal in other proteins (71,72) and may be required to target FANCD2 to nuclear foci. Several other proteins involved in DNA repair also form nuclear foci after DNA damage. These include BRCA1, BRCA2, Rad51 and p95/NBS (73–76). Indeed, the nuclear foci formed by FANCD2 upon DNA damage co-localize with the foci formed by the BRCA1 protein, mutated in familial forms of breast cancer (70,77). This finding indicates that FANCD2 may function in the same DNA damage response pathway(s) as BRCA1 (Fig. 1) (75,77–79). Indeed, BRCA1 mutant cells are very sensitive to DNA cross-linkers and show a spectrum of sensitivity to DNA damaging agents that is very similar to FA cells (70,79). In male meiosis, FANCD2 also has a location similar to BRCA1 (70,73). FANCD2 antibodies stain meiotic chromosomes at synaptonemal complexes. Synaptonemal complexes represent sites of meiotic recombination (80) and defects of homologous recombination (HR) for DNA repair have been found in both BRCA1 and BRCA2 mutant cells (78,79). This raises the question whether the FA pathway could function directly in recombinational DNA repair or act to modulate such a process. Indeed, mutations in genes involved in HR result in sensitivity to ICL in multiple species, consistent with the FA cellular phenotype.

At least two additional FA proteins, FANCB and FANCD1, have not yet been cloned. The FA protein complex (A/C/E/F/G) fails to form in FA-B cells, suggesting that the FANCB product may be required for the assembly or stability of the complex (56). In FA-D1 cells, the FA protein complex forms normally (62) and the FANCD2 is normally mono-ubiquitinated in response to DNA damage. Taken together, these data suggest that the FANCD1 protein may function downstream or independently of the pathway. There are several features of the model which remain unresolved. First, it is not known whether the FA protein complex is a constitutive multisubunit complex or whether it assembles in stages. For instance, recent studies demonstrate that the subunits of the complex (A, C, E, F and G) have variable affinities for each other (60). The A protein strongly interacts with G, and G strongly interacts with F (59,60). Accordingly, smaller complexes, consisting of fewer subunits, may assemble in stages and may have discrete functions in the cell, independent of the large multisubunit structure. Secondly, it is possible that other protein intermediates exist between the FA protein complex and the FANCD2 protein. For instance, it is not known whether the FA protein complex has intrinsic ubiquitin ligase activity or whether it functions upstream of a ubiquitin ligase. Along these lines, the FANCD2 protein does not form a stable interaction with the FA protein complex, suggesting that it may interact with the complex indirectly, through other protein intermediates or following posttranslational modifications of its subunits. Thirdly, in addition to FANCD2, there may be other downstream substrates in the FANCD2 pathway. For instance, other (unknown) proteins may be mono-ubiquitinated by the multisubunit FA complex (81). However, since FA-D2 patients have the same clinical and cellular phenotype as other FA complementation groups, FANCD2 may be the sole substrate of the FA protein complex. Fourthly, although the mono-ubiquitinated isoform of the FANCD2 protein co-localizes and co-immunoprecipitates with BRCA1, the precise nature of this interaction remains unknown. Recent studies have demonstrated that BRCA1 has ubiquitin ligase activity (82,83) and it is therefore a prime candidate to be involved in FANCD2 ubiquitination. FANCD2 and BRCA1 do not interact directly, as judged by two-hybrid interaction studies (unpublished data), suggesting that this interaction may be indirect and mediated by other protein intermediates (for example, other FANC proteins). MAMMALIAN ICL REPAIR AND FA Interstrand DNA cross-links represent an important class of DNA damage, but little is known about the repair of these lesions in mammals. Several DNA cross-linking drugs are currently in clinical use including mitomycin C, cisplatin, cyclophosphamide and psoralen + UVA irradiation. ICL are unique in that both strands of the DNA are simultaneously damaged and no undamaged strand is available as a template for repair (Fig. 2). Furthermore, an ICL represents an absolute block to both transcription and DNA replication. Evidence exists for both HR-dependent and -independent mechanisms of ICL repair in mammalian cells (Fig. 2) (84). Abnormalities of both of these pathways have been reported in FA cells, but no


Figure 2. Basic considerations regarding ICL repair. (A) ICL occurred after replication and the homologous region on sister chromatid has been generated. HR between sister chromatids produces an error free repair (top). If repair does not occur, mitosis proceeds normally, but one of the daughter cells inherits an unrepaired ICL (bottom). (B) ICL was generated before replication and hence a homologous sister chromatid is not available for recombinational repair. (C) Possible mechanisms for repair of pre-replication ICLs. Non-homologous end joining can remove the ICL, but will produce mutations (top). Excision repair and/or lesion bypass may remove the ICL, but also will be error-prone (middle). HR between homologues chromosomes (autosomes and X-chromosomes in females) could result in error-free ICL repair (bottom). (D) An unrepaired ICL generates a mandatory chromatid-break during mitosis.

definitive defects have been found. Using plasmid substrates, it was discovered that FA cell nuclear extracts had reduced fidelity of NHEJ (85,86). Another study reported a marked increase in HR in FA cell nuclear extracts, again using plasmid substrates (87). Overall, the recent observations regarding FANCD2 localization to BRCA1 foci (see above) are in better support of a role of FA in HR-mediated repair. Only few studies on mutagenesis in FA cells have been reported. When the HPGRT locus was used as a mutation target, FA cells were found to be ‘hypo-mutable’, i.e. produce fewer clones with mutations at this locus (88). In addition, an abnormal spectrum of mutations was found with a notable increase in deletions (89,90). DNA repair by recombination requires a homologous DNA sequence in a different location than the DNA damage. Possible templates for recombination include related sequences on the same chromosome (intrachromosomal recombination), sister chromatids and sister chromosomes (homologues). Sister chromatid recombination would be well suited to repair ICL introduced after DNA replication. Indeed, double-strand breaks introduced by IR can be repaired by this mechanism (91) and ICL treatment can induce increased frequencies of sister chromatid exchange (92). In contrast to

Bloom’s syndrome cells, however, FA cells do not differ significantly from controls in sister chromatid exchange rates (93,94). A recent study showed that most ICL introduced after DNA replication are not repaired during the G2 phase of the cell cycle and do not induce a G2/M arrest or chromosome breakage (95). Instead, such lesions were only sensed during the next S-phase of the next cell cycle. Thus, mammalian cells recognize and repair ICL in the context of DNA replication and do not significantly use sister chromatid recombination during G2 to repair the lesion. Consistent with this observation, it is known that ICL-induced cytogenetic damage is of the ‘chromatid type’, i.e. requires prior DNA replication. Thus, the only undamaged template available for recombinational repair of ICL incurred prior to completion of DNA replication are the homologous chromosomes (Fig. 2). Indeed, recombination between homologues has recently been reported as a mechanism for double-strand break repair in mammalian cells (96). Because ICL are normally repaired during S-phase and because FA cells are hypersensitive to ICL, it can be hypothesized that the FA proteins may function in an S-phase-specific DNA repair pathway. Consistent with this notion, the FA pathway (i.e. mono-ubiquitination of the FANCD2 protein) is activated


specifically during the S-phase of the cell cycle (A.D’Andrea and M.Grompe, unpublished observation). Direct or indirect role in DNA repair? The typical chromosome breakage observed in FA cells as well as the cancer predisposition in this syndrome leave little doubt that the FA proteins are involved in cellular responses to DNA damage, especially ICL. However, the role of FA proteins in DNA repair could either be direct or indirect. FA proteins themselves may sense ICL or mediate ICL repair by either HR or HR-independent mechanisms. This ‘direct involvement hypothesis’ is supported by a recent report demonstrating direct binding of FA proteins along with α-spectrin to psoralencrosslinked DNA (64,97,98). Alternatively, the FA proteins may only be indirectly involved in ICL repair, for example by triggering a cell cycle checkpoint. In support of this hypothesis it has recently been shown that FA cells display radiationresistant DNA synthesis after ICL treatment (99,100). These findings indicate that FA proteins may mediate an S-phase cell cycle checkpoint. Failure of this checkpoint would result in inefficient ICL repair and a prolonged ‘G2/M’ phase of the cell cycle. It is of course also possible that the FA pathway does both, i.e. repairs DNA damage as well as modulates cell cycle responses to DNA lesions. In the future, the identification of the other genes in the FA pathway (i.e. the as yet uncloned FANCB and FANCD1 genes) or identification of FANCD2 binding proteins may further elucidate the functions of the FA pathway in DNA repair. ACKNOWLEDGEMENTS This work was supported by NHLBI program project grant nos 1PO1HL48546 (M.G.) and R01HL52725 (A.D’A.). REFERENCES 1. Fanconi, G. (1967) Familial constitutional panmyelocytopathy, Fanconi’s anemia (F.A.) I. Clinical aspects. Semin. Hematol., 4, 233–240. 2. Alter, B.P. (1995) Hematologic abnormalities in Fanconi anemia. Blood, 85, 1148–1149. 3. Butturini, A., Gale, R.P., Verlander, P.C., Adler-Brecher, B., Gillio, A.P. and Auerbach, A.D. (1994) Hematologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study. Blood, 84, 1650–1655. 4. Alter, B.P. (1996) Fanconi’s anemia and malignancies. Am. J. Hematol., 53, 99–110. 5. Alter, B.P. and Tenner, M.S. (1994) Brain tumors in patients with Fanconi’s anemia. Arch. Pediatr. Adolesc. Med., 148, 661–663. 6. Alter, B.P. (1993) Fanconi’s anaemia and its variability. Br. J. Haematol., 85, 9–14. 7. Sasaki, M.S. and Tonomura, A. (1973) A high susceptibility of Fanconi’s anemia to chromosome breakage by DNA cross-linking agents. Cancer Res., 33, 1829–1836. 8. Auerbach, A.D. and Wolman, S.R. (1976) Susceptibility of Fanconi’s anaemia fibroblasts to chromosome damage by carcinogens. Nature, 261, 494–496. 9. Auerbach, A.D. (1993) Fanconi anemia diagnosis and the diepoxybutane (DEB) test. Exp. Hematol., 21, 731–733. 10. Gluckman, E. (1990) Radiosensitivity in Fanconi anemia: application to the conditioning for bone marrow transplantation. Radiother. Oncol., 1, 88–93. 11. Noll, M., Bateman, R.L., D’Andrea, A.D. and Grompe, M. (2001) Preclinical protocol for in vivo selection of hematopoietic stem cells corrected by gene therapy in Fanconi anemia group C. Mol. Ther., 3, 14–23.

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