REVIEWS

THE FANCONI ANAEMIA/BRCA PATHWAY

Alan D. DAndrea* and Markus Grompe
Fanconi anaemia (FA) is a rare genetic cancer-susceptibility syndrome that is characterized by congenital abnormalities, bone-marrow failure and cellular sensitivity to DNA crosslinking agents. Seven FA-associated genes have recently been cloned, and their products were found to interact with well-known DNA-damage-response proteins, including BRCA1, ATM and NBS1. The FA proteins could therefore be involved in the cell-cycle checkpoint and DNA-repair pathways. Recent studies implicate the FA proteins in the process of repairing chromosome defects that occur during homologous recombination, and disruption of the FA genes results in chromosome instability a common feature of many human cancers.
PANCYTOPAENIA

Greatly decreased levels or absence of primary haematopoietic cells in the bone marrow. In this state, there are decreased numbers of lymphocyte, red blood cell and platelet progenitors.

*Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA. Department of Molecular and Medical Genetics, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road L103, Portland, Oregon 97239, USA. Correspondence to A.D.D. e-mail: alan_dandrea @dfci.harvard.edu
doi:10.1038/nrc970

Fanconi anaemia (FA) is a rare autosomal recessive disease that is characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility 1,2. Children with FA frequently develop PANCYTOPAENIA during the first decade of life, and death often results from complications of bone-marrow failure. Although FA has an incidence of less than 1 per 100,000 live births, a molecular understanding of the FA pathway might have relevance to other types of cancer. The recent cloning of seven FA genes has improved the prospects for diagnosis and therapy, and has elucidated the mechanisms of chromosome instability in this cancer. The clinical features of FA have been extensively reviewed. These include skeletal abnormalities (that is, thumb or limb abnormalities) and abnormal skin pigmentation (that is, hypopigmentation or caf au lait spots) 3. Other organ systems that are involved include the cardiac, renal and gastrointestinal systems, and individuals with FA have decreased fertility. Accordingly, the FA genes are necessary for the normal development of several organ systems. The haematological consequences of FA often develop in the first few years of life. Anaemia and thrombocytopaenia (decreased platelet count) often proceed the neutropaenia (decreased white-cell count). FA patients also develop clonal chromosomal abnormalities in the bone-marrow progenitor cells

(that is, chromosome 1 or 2 abnormalities or monosomy 7), and these can develop into myelodysplasia or acute myeloblastic leukaemia (AML). FA is considered to be a genetic model system for the study of myelodysplastic syndrome or myeloid leukaemia. FA patients are predisposed to many types of cancer4. Although AML is the most common cancer in these patients, older patients can develop squamouscell carcinomas of the head and neck or gynaecological system. Recent studies have indicated a high incidence of oesophageal cancer in FA patients who have received a bone-marrow transplant5. FA like other rare, inherited cancer-susceptibility syndromes has provided important insights into the genetic basis of cancer in the general population. The FA-associated gene products along with BRCA1 and BRCA2 have been found to function in a common pathway that regulates the cellular response to DNA damage. Inherited defects in this pathway underlie the chromosomal instability and drug sensitivity of at least a subset of solid tumours in the general (non-FA) population. Strikingly, the FA/BRCA pathway has numerous molecular interactions with other proteins that are known to mediate checkpoint responses, such as ataxia telangiect-asia mutated (ATM) and Nijmegen breakage syndrome 1 (NBS1), as well as the DNA-repair response protein RAD51.

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abnormalities that occur during metaphase. Crosslinker hypersensitivity can result in apoptosis or growth arrest, depending on the cell type. FA cells also have more modest hypersensitivity to other DNA-damaging agents, such as ionizing irradiation and oxygen radicals. A second phenotype of FA cells is an increase in the proportion of cells with 4N DNA content7,8. This indicates that a G2/M or late S-phase delay occurs in the cell cycle9. The increase of cells with 4N DNA content can occur spontaneously in some FA cells, but becomes much more pronounced after treatment with crosslinking agents. Some reports indicate that FA cells have primary defects in signalling and apoptosis induction pathways. The relationship of these other phenotypes to DNA damage is less clear. The hypersensitivity of FA cells to DNA crosslinking agents, such as diepoxybutane and mitomycin C, provides a convenient diagnostic test for the disease10. The diepoxybutane chromosome breakage test, which is performed on peripheral-blood lymphocytes, remains the clinically certified diagnostic tool for FA. The diepoxybutane test can also be performed on primary fibroblast cultures. As several FA genes have been cloned, direct sequencing of the genes can be used in prenatal diagnosis. More recently, the ability to complement cells from FA patients with retroviral vectors that encode FA-associated genes, along with the development of immunoblots for FA proteins, have improved the ability to diagnose this disease and to determine the subtype1113.
Treatment of FA

Summary Fanconi anaemia (FA) is an autosomal recessive disease that is characterized by developmental abnormalities, cancer susceptibility and cellular sensitivity to crosslinking agents. The seven cloned FA proteins interact in a common pathway. Five FA proteins (A, C, E, F and G) regulate the activation, via monoubiquitylation, of FANCD2. Activated FANCD2 is targeted to BRCA1 nuclear foci. The FANCD1 gene is BRCA2. The FA/BRCA pathway seems to regulate DNA repair by homologous recombination. Ionizing radiation activates the ATM-dependent phosphorylation of FANCD2, resulting in an intra-S checkpoint response. FANCD2 interacts with the MRE11NBS1RAD50 complex in the repair of DNA crosslinks. Somatic inactivation of the FA/BRCA pathway accounts for the chromosomal instability of some cancers in the general population. Mouse models for FA subtypes A, C, D1, D2 and G have been generated. DNA crosslink repair, which is defective in cells from FA patients, requires S-phase arrest and homologous recombination repair.

Cellular phenotype of FA

Many phenotypes have been reported to characterize cells from patients with FA6 (BOX 1). The most consistent of these phenotypes is hypersensitivity to agents that produce interstrand DNA crosslinks, such as mitomycin C, diepoxybutane and cisplatin. After treatment with crosslinking agents, FA cells have increased chromosome breakage, RADIAL CHROMOSOMES and other cytogenetic

Box 1 | Phenotypic features of Fanconi anaemia cells Several studies have indicated various phenotypic features of Fanconi anaemia (FA) cells. The most consistent of these features is the generation of chromosome breaks and radial forms in response to crosslinking agents. These phenotypic features have recently been reviewed6: Sensitivity to crosslinking agents Prolongation of the late S phase and G2 phase of the cell cycle Sensitivity to oxygen Grow poorly in the presence of oxygen Overproduction of oxygen radicals Deficient oxygen radical defence Deficiency in superoxide dismutase Sensitivity to ionizing radiation (G2-phase specific) Overproduction of tumour necrosis factor- Defects in DNA Repair Accumulation of DNA adducts Defective in repair of DNA crosslinks Genomic instability Spontaneous chromosome breakage Hypermutable (by deletion mechanism) Increased apoptosis Defective p53 induction Stem-cell defects Decreased colony growth in vitro Decreased gonad stem-cell survival

The treatment of FA is largely directed towards the complications of bone-marrow failure. Some FA patients respond initially to haematopoietic growth factors, such as erythropoietin and granulocytemacrophage colonystimulating factor14. Others respond to androgen therapy, although long-term androgen exposure might result in liver disease. The best treatment for pancytopaenia that is associated with FA is matched allogeneic bone-marrow transplantation. In the absence of a histocompatible matched sibling, unrelated donor transplants15 and cordblood transplants1618 are also routinely performed. In addition, new approaches involve pre-implantation genetic diagnosis19 and gene therapy20,21 for select patients with FA. FA patients who survive bone-marrow disease remain prone to malignancies that usually occur in the second or third decade of life. Early surgical excision of these tumours is crucial, as FA patients experience toxic side effects after chemotherapy or radiation therapy.
Genetics

Cell-fusion experiments have shown the existence of at least eight separate FA COMPLEMENTATION GROUPS. These are FA-A, B, C, D1, D2, E, F and G. So far, seven FA genes have been cloned2226. The genes are not clustered, but are instead widely dispersed throughout the genome (TABLE 1). In most cases, these genes were cloned by functional complementation of mitomycin-C-sensitive cells with cDNAs (FANCC, A, G, F and E, in chronological order). Two of the genes were isolated by positional cloning approaches (FANCA and

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require expression of FANCG37. FANCA and FANCC failed to co-immunoprecipitate from lysates of FA cells that are derived from complementation groups B, E and F 38,39, indicating that several FA proteins are probably required to form the complex. The direct binding interactions among some FA proteins were further confirmed by two-hybrid analyses40 (FIG. 1). Loss of one FA protein, such as FANCA or FANCG, resulted in instability of the FA complex and a decreased half-life of newly synthesized FA proteins41. The FA protein complex was, however, detected in cell lines derived from other FA complementation groups, such as the D1 and D2 groups. These results indicated that the protein products of the D1 and D2 genes are not required for protein-complex assembly. The FA complex seems to have some chromatin binding activity, which is enhanced by exposure to crosslinking agents42. Recent evidence indicates that the FANCE protein might interact with FANCD2 (REF. 43), although FANCD2 is not an integral component of the FA complex 44. Although the FA proteins form a multisubunit complex, the function of the complex remains unknown. More research is required to determine whether the complex plays a direct role in sensing DNA damage, repairing DNA damage or stabilizing chromosome structures. None of the FA proteins in the complex has an obvious catalytic domain and, so far, no enzymatic activity has been associated with the purified complex. The FA complex might be an indirect upstream regulator or sensor in the DNA-damage response.

Table 1 | Complementation groups of Fanconi anemia

Subtype A B C D1 D2 E F G FA patients, (estimated %) 65% Rare 15% Rare Rare Rare Rare 10% Chromosome location 16q24.3 13q1213 9q22.3 13q1213 3p25.3 6p2122 11p15 9p13 Protein products (kD) 163 380 (BRCA2) 63 380 (BRCA2) 155,162 60 42 68 (XRCC9)

RADIAL CHROMOSOMES

Abnormal chromosome structures that result from pairing of homologous or nonhomologous metaphase chromosomes. These structures are observed in chromosome spreads prepared from cells with underlying chromosome instability, such as cells from patients with Fanconi anaemia, Bloom syndrome and ataxia telangiectasia.
COMPLEMENTATION GROUPS

FANCD2)27,28. Complementation groups A (~65%), C (15%) and G (10%) account for most FA patients in most populations. Some ethnic groups have a higher occurrence of specific mutations that account for most cases in these populations. The FANCC IVS4+4 AT allele is prevalent among Ashkenazi Jews, with a carrier frequency of about 1 in 150 (REFS 29,30). Similarly, FANCA mutations are common in the Afrikaners of South Africa, due to a FOUNDER EFFECT. Overall, FA is genetically heterogeneous, and many mutations have been found in each complementation group. There is only a modest correlation between phenotype and subtype. Hearing deficits, for example, are found more commonly in patients with mutations in FA-G. Also, patients with FA subtypes C or G tend to have earlier onset of bone-marrow failure and haematological malignancies, compared with patients who have mutations in FA-A31. Nonetheless, the complete spectrum of phenotypic variation can be found within single complementation groups, and certain phenotypes can be correlated strongly with specific mutations. For example, patients that are homozygous for the FANCC IVS4+4 AT mutation develop severe FA, with a high frequency of birth defects and early onset of the anaemia32. By contrast, patients that are homozygous for the FANCC 322G mutation have comparatively few birth defects and develop anaemia later in life32.
FA protein interactions

Nucleus

DNA damage/ replication

DNA repair

BRCA1 FA complex G F D2 K561 A C E BRCA2 RAD51

These subsets of Fanconi anaemia were established by somatic-cell fusion analysis of cells derived from patients with the disease. Two cell lines are members of two different complementation groups if the fused line has complemented or restored its normal cellular phenotype (that is, mitomycin C resistance).
FOUNDER EFFECT

The first six FA genes that were cloned have no sequence similarity to each other or to other known DNA-repair genes. Of these genes, only FANCD2 has homologues in other eukaryotic species, including Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Fugu and zebrafish28,33. This indicates that FANCD2 has an important conserved cellular function. As the biallelic disruption of any one of these six FA genes results in similar cellular and organismal phenotypes, investigators proposed that the six proteins cooperated in a cellular pathway34,35. A multisubunit nuclear complex. Biochemical studies indicated that the FANCA and FANCC are part of the same complex36, although they have not been shown to interact directly in vitro. This interaction might require additional proteins, as it has been shown to

RA

RAD

D5

D2 K561 Ub

DNA-repair foci

High frequency of a rare genetic mutation in a population. The mutation originally present in a founder individual becomes prevalent by inbreeding.

Figure 1 | The Fanconi anaemia/BRCA pathway. Several FA proteins, including A, C, E, F and G, form a constitutive complex in the nucleus of normal human cells. In response to DNA damage, or during the S phase of the cell cycle, this complex mediates the monoubiquitylation (Ub) of FANCD2 at lysine 561 (K561). Activated FANCD2, in turn, is translocated to chromatin and DNA-repair foci. These foci contain the BRCA1 protein and the BRCA2FANCD1 protein complex. BRCA2/FANCD1 is known to bind directly to RAD51 and to DNA, and to participate in homology-directed DNA repair. Taken together, the model indicates that the FA/BRCA pathway regulates DNA repair.

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The FANCD2 protein. The FANCD2 protein seems to function at a downstream point in the FA pathway45 (FIG. 1). Initial studies indicated that the FANCD2 protein exists in two cellular isoforms: FANCD2-S (the primary unmodified translation product) and FANCD2-L (the monoubiquitylated isoform). Importantly, the FANCD2-L isoform is only observed in wild-type or complemented FA cells that are resistant to mitomycin C. This finding indicates that the FA complex and all of the FA subunits therein are required for the conversion of FANCD2-S to FANCD2-L. Mass spectrometric analysis showed that this modification is a monoubiquitylation of FANCD2 on Lys561 (K561). Mutation of this lysine residue to arginine ablates monoubiquitylation, indicating that other lysine residues in the protein cannot substitute as sites of ubiquitin attachment. This lysine (K561) is also conserved in many lower eukaryotic FANCD2 homologues28, further supporting its crucial function. The conversion of FANCD2-S to FANCD2-L is required for DNA-damage-inducible changes in FANCD2 localization. Monoubiquitylated FANCD2 is targeted to subnuclear foci, where it co-localizes with BRCA1 (REF. 45) and RAD51 (REF. 46). FANCD2 foci do not form in FA cells of subtypes A, B, C, E, F or G, but do assemble in complemented cells. Importantly, FANCD2 monoubiquitylation and targeting to foci is induced by either DNA-damaging agents such as ionizing radiation, ultraviolet light or mitomycin C, or during the S phase of the cell cycle. This damage-inducible response indicates that the activated FANCD2 protein might interact with other DNA-damage-response proteins that are known to be associated with BRCA1 foci47. Interaction with BRCA proteins. Several lines of evidence have indicated that FANCD1 might be related to BRCA1 and BRCA2. Disruption of BRCA1 results in loss of DNA-damage-inducible FANCD2-containing subnuclear foci. BRCA1 might therefore regulate the assembly of these foci45. Consistent with this, the BRCA1 protein contains a RING-FINGER DOMAIN, which is commonly observed in some E3 ubiquitin ligases. BRCA1 might therefore ubiquitylate FANCD2 in vivo48. BRCA1/ or BRCA2 / cells also show mitomycin C hypersensitivity and chromosome instability49, similar to the defects observed in FA cells. Functional complementation of BRCA2 / cells with wild-type mouse Brca2 restores mitomycin C resistance50, and targeted inactivation of the Brca2 gene (by disrupting the region that encodes the carboxyl terminus but not the amino terminus) results in viable mice with a FA-like phenotype. Features of this phenotype include small size, skeletal defects, hypogonadism, cancer susceptibility, chromosome instability and mitomycin C hypersensitivity51. To investigate the relationship between BRCA genes and FA, the BRCA1 and BRCA2 genes were sequenced in cells from FA-B, FA-D1 and unassigned FA patients52. Although no BRCA1 mutations were detected, biallelic mutations in BRCA2 were observed in these cells. The most common BRCA2 mutations observed were frameshift mutations in the 3 region of the gene, which would be predicted to cause truncations of the carboxyl terminus. Indeed, BRCA2 proteins with carboxy-terminal truncations were detected in cell lines derived from some patients with FA. These mutant BRCA2 proteins might have partial activity. Functional complementation of FA-D1 fibroblasts with wild-type BRCA2 cDNA restored mitomycin C resistance, indicating that FANCD1 is identical to BRCA2. Paradoxically, cell lines derived from a patient in the FA-B complementation group also contained two mutant BRCA2 alleles. One mutant allele contained a frameshift mutation in exon 11, and the second allele contained a polymorphic stop codon in exon 27. The FA-D1 and FA-B cells have biallelic mutations in the same gene (BRCA2), indicating intragenic or interallelic complementation. Alternatively, the protein product of some mutant BRCA2 alleles might have dominant activity in vivo, accounting, at least in part, for these discrepancies.
Regulation of the FA/BRCA pathway

RING-FINGER DOMAIN

A distinct zinc-binding domain that is present in many E3 ubiquitin ligases. The RING finger binds two zinc atoms, with each atom ligated in a tetrahelix by four cysteines, or three cysteines and one histidine.

Individuals with biallelic BRCA2 mutations share clinical features with other FA patients. These include congenital abnormalities, abnormal skin pigmentation, bone-marrow failure and cellular sensitivity to mitomycin C. These similarities indicate that BRCA2 and other FA proteins cooperate in a common DNA-damage-response pathway (FIG. 1). According to this model, DNA damage activates the monoubiquitylation of FANCD2, which targets it to DNA-repair foci that contain BRCA1 and BRCA2. Previous studies have indicated that FANCD2 is not monoubiquitylated in mutant FA-B cells, whereas it is in mutant FA-D1 cells. BRCA2 might therefore function upstream in the pathway, by promoting FA-complex assembly and FANCD2 activation, and/or downstream in the pathway, by transducing signals from FA proteins to RAD51 and the homologous recombination machinery. Formation of RAD51-containing foci seems to be reduced in some FA cell lines, such as mutant FA-D1 fibroblasts53. This indicates that the FA pathway might be required for the organization of RAD51 into functional DNA-repair units. The precise molecular function(s) of BRCA1 and BRCA2 in this pathway remain to be elucidated. A less likely scenario is that BRCA2 functions independently of the FA pathway, and that disruption of BRCA2 phenocopies the disruption of the FA-associated genes. Patients with FA that also have BRCA2 mutations seem to have a more severe clinical phenotype, with earlier onset of myeloid leukaemia and other malignancies. Whether FA patients with BRCA2 mutations have other unique developmental abnormalities has not been determined, due to the small number of patients. Other unresolved questions arise from the model that is proposed in FIG. 1. First, little is know about the exact molecular mechanism of FANCD2 monoubiquitylation. Although the FA complex is required for this modification, none of the known FA proteins have a catalytic domain of a ubiquitin ligase. Ubiquitin ligases

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FANCD2-S during the mitotic phase of the cell cycle46. Deubiquitylating enzymes are a relatively new family of regulatory enzymes, and their specific substrates are unknown56.
FA and ATM kinase

Ionizing radiation

Mitomycin C

DSB

DNA damage

ATM

FA

D2 S222

D2 S222 P

D2 K561

D2 K561 Ub DNA repair

S-phase arrest

Figure 2 | Interaction of the FA/BRCA pathway with the ATM kinase. The FANCD2 protein functions at the intersection of two signalling pathways, and undergoes two independent types of post-translational modification. In response to ionizing-radiation-mediated double-strand breaks (DSB), the ATM kinase phosphorylates (P) FANCD2 on serine 222 (S222), leading to activation of an S-phase checkpoint. In response to mitomycin C treatment or exposure to ionizing radiation, the FA complex mediates the monoubiquitylation (Ub) of FANCD2 at lysine 561 (K561), thereby activating a DNA-repair response.

HECT DOMAIN

(Homologous to E6-AP carboxyl terminus domain). A distinct domain that is characteristic of another subclass of E3 ubiquitin ligases. Mammalian HECT E3s include E6-AP, which targets p53 for ubiquitylation in the presence of human papillomavirus E6.

often contain ring fingers that are homologous to E6AP carboxyl terminus (HECT) DOMAINS. As BRCA1 colocalizes with activated FANCD2, BRCA1 or BARD1 (BRCA1-associated ring finger E3 ubiquitin ligase) could serve this function. Consistent with this idea, the BRCA1 protein interacts with FANCA one of the subunits of the FA-protein complex54. Alternatively, the RAD6RAD18 complex, which monoubiquitylates histones 2A and 2B, might function as a ubiquitin ligase for FANCD2. It will be important to determine whether BRCA1 or BARD1 can monoubiquitylate FANCD2 in vitro, and whether cells that are deficient in these E3 ring-finger ligases have decreased levels of FANCD2-L. Recent studies indicate that monoubiquitylation is crucial for the association of FANCD2-L with damaged chromatin55. A mutant form of FANCD2 that fails to undergo monoubiquitylation (FANCD2-K561R) remains in the soluble nuclear fraction and fails to bind chromatin. The molecular basis for this targeting remains unclear, but indicates that some sort of monoubiquitin receptor could be present at sites of DNA repair. Finally, several other aspects of the FA pathway remain unclear. First, additional regulatory enzymes modulate the pathway. For instance, ubiquitylation events are often preceded by phosphorylation events. As FANCD2 is monoubiquitylated following DNA damage or during S phase of the cell cycle, different kinases might phosphorylate FANCD2 near lysine 561 to activate its monoubiquitylation. Also, precursor/product analysis reveals that the monoubiquitylated form of FANCD2 is recycled back to the unubiquitylated isoform. So, a deubiquitylating enzyme could function to convert FANCD2-L to

In addition to their mitomycin C sensitivity, FA cells also show hypersensitivity to ionizing radiation57,58, indicating that the FA pathway might interact with the kinase ATM. Ataxia telangiectasia (AT) is another autosomal recessive human disease that results in spontaneous chromosome breakage and haematological cancers59. Unlike FA patients, AT patients have immunodeficiency and progressive cerebellar neural degeneration. AT cells are hypersensitive to ionizing radiation but are not hypersensitive to crosslinking agents. The ATM gene encodes an ionizing-radiationactivated protein kinase that phosphorylates and activates proteins that are involved in cell-cycle checkpoint responses, including p53 (REFS 60,61), CHK2, Nijmegen breakage syndrome (NBS1)62,63 and BRCA1 (REF. 64). Many of these proteins cooperate in the ionizing-radiation-activated S-phase checkpoint response. Biallelic loss of ATM or its substrates results in a defect in the ionizing-radiation-activated S-phase checkpoint. Interestingly, cells that are derived from patients with the FA-D2 subtype, but not from other subtypes, have a similar defect in the ionizing-radiation-inducible Sphase checkpoint, indicating possible molecular interactions between ATM and FANCD2 (REF. 65). Further studies indicated that, in response to cellular exposure to ionizing radiation, the ATM directly phosphorylates FANCD2 on serine 222, as well as on other sites (FIG. 2), and this is required for the establishment of the S-phase checkpoint. A mutant form of FANCD2 (FANCD2-S222A) that is not phosphorylated by ATM fails to activate the checkpoint in transformed human fibroblasts. Transfection of the mutant FANCD2 (S222A) into wild-type cells induces an S-phase checkpoint defect, which is characterized by persistent DNA synthesis even after cellular exposure to ionizing radiation (K. Nakanishi and A. DAndrea, unpublished observations). This defect in the intra-S-phase checkpoint has been observed in human SV-40 transformed FA-D2 cell lines and has not been confirmed in mutant FA-D2 primary fibroblasts. It is not clear how the ATM-dependent phosphorylation of FANCD2 establishes the ionizing-radiation-inducible S-phase checkpoint, but there are several possible models. First, other ATM-activated proteins, such as NBS1 and BRCA1, might physically interact with phosphorylated FANCD2 in a checkpoint protein complex. Such a multisubunit complex could act as a sensor of DNA damage, and might bind and locally inhibit DNA replication. Second, the ATM-dependent phosphorylation of FANCD2 might function as an amplifier of the checkpoint signal. Since the S-phase checkpoint occurs in cells that express the K561R mutant of FANCD2, monoubiquitylation of this protein and foci formation are not required for this amplifier activity.

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checkpoint might allow cells with unrepaired chromosomes to progress through the cell cycle, ultimately leading to cell death. Several aspects of the interaction between ATM and the FA pathway remain unresolved. First, FA cells that are derived from other (non-D2) subtypes have mild hypersensitivity to ionizing radiation, and the mechanism for the phenotype is unclear. Perhaps some DNA damage by ionizing radiation mimics the damage of mitomycin C. Second, FANCD2 is phosphorylated in ATM/ cells that have been exposed to genotoxic agents such as mitomycin C or hydroxyurea, indicating a possible involvement of other ATM-like kinases such as ATR or CHEK2 (REF. 67). So far, the possible role of such kinases in FANCD2 activation is unknown. Third, the relative contribution of FANCD2 and other ATM substrates to the S-phase checkpoint response remains unknown.
The NBS1MRE11RAD50 complex

ATM S-phase arrest P S222 D2 K561 Ub FA complex P NBS1 MRE11 RAD50 Nuclear foci

DNA damage

DNA repair

Figure 3 | Interaction of the FA/BRCA pathway with the MRE11 complex (NBSMRE11RAD50). In response to ionizing radiation, ATM phosphorylates (P) the NBS1 protein. Phosphorylation of NBS1 is required for FANCD2 phosphorylation at serine 222 (S222), and ultimately for activation of the intra-S-phase checkpoint response. The monoubiquitylated (Ub) isoform of FANCD2 also co-localizes with the NBSMRE11RAD50 complex in DNA-damageinducible subnuclear foci, which are required for DNA repair by homologous recombination.

So FANCD2 seems to function at the intersection of two signalling pathways (FIG. 2). In response to ionizing radiation, ATM phosphorylates FANCD2 on serine 222, resulting in the establishment of an S-phase checkpoint. In response to mitomycin C or ionizing radiation, the FA-protein complex and BRCA1 cooperate to regulate the monoubiquitylation of FANCD2 on lysine 561, resulting in mitomycin C resistance45. These two post-translational modifications and functions of FANCD2 can be dissociated. First, ionizing-radiation-dependent, ATM-mediated phosphorylation of FANCD2 is independent of FANCD2 monoubiquitylation. Following exposure to ionizing radiation, FANCD2 phosphorylation occurs in FA cells of subtypes A, C, G and F, although FANCD2 is not monoubiquitylated in these cells. Also, the K561R mutant of FANCD2 is phosphorylated following ionizing radiation and corrects the S-phase checkpoint, even though this protein is not monoubiquitylated. Second, monoubiquitylation of FANCD2 is independent of FANCD2 phosphorylation on serine 222. FANCD2 monoubiquitylation and foci formation occurs in ATM/ cells. Although phosphorylation on serine 222 is not essential for monoubiquitylation, phosphorylation of this residue, or some other residue, might enhance FANCD2 monoubiquitylation. Phosphorylation primes the polyubiquitylation of many protein substrates, although its effect on protein monoubiquitylation remains unknown. The sensitivity of FA cells to crosslinking agents might also be caused by improper checkpoint regulation during S phase. Like FANCD2 / cells, BRCA2/FANCD1deficient Chinese hamster ovary (CHO) cells50, as well as BRCA1-deficient cells66, undergo radioresistant DNA synthesis. Taken together, a complex of ATM-activated FANCD2, BRCA1 and BRCA2/FANCD1 might regulate the S-phase checkpoint. In FA cells, disruption of the

Nijmegen breakage syndrome (NBS) is another autosomal-recessive chromosome-breakage disease68. It is similar to AT in that it is characterized by immunodeficiency and predisposition to lymphoma. A small number of patients have FA-like features, such as bone-marrow failure and myeloid leukaemia. NBS results from biallelic mutation of the NBS1 gene. The NBS1 protein is phosphorylated by ATM and normally forms a complex with MRE11 and RAD50 (the MRE11 complex). Like ATM, the MRE11 complex is a crucial upstream regulator of checkpoint responses and DNA-repair responses in all eukaryotic cells67,69. NBS cells have biallelic mutations in NBS1. In mammalian cells, the MRE11 complex is required for DNA repair and for normal S-phase checkpoint function. In yeast, the MRE11 complex is required for normal double-strand-break repair, either through homologous recombination or non-homologous end-joining (NHEJ) mechanisms70. The MRE11 complex assembles with BRCA1 in nuclear foci following DNA damage71, indicating that the complex could regulate homologous recombination repair. ATM phosphorylates NBS1 on serine 343 and activates the S-phase checkpoint62,63. There are some phenotypic similarities between patients with FA and those with NBS. For instance, some NBS patients have bone-marrow failure72 and other features of FA. Also, NBS cells show mitomycin C hypersensitivity, although this sensitivity is not as severe as it is in cells that are taken from patients with FA. A possible interaction between FA and NBS was indicated by the identification of a patient with atypical FA, which is characterized by diepoxybutaneinduced chromosome breakage, who had biallelic mutations in the NBS1 gene 73. Based on these similarities, the potential interaction between the NBS and FA pathways were investigated73 (FIG. 3). In response to mitomycin-C-mediated DNA damage, the NBS1 protein and the activated form of FANCD2 (FANCD2-L) co-localized in subnuclear foci. Preventing NBS1/FANCD2 foci formation, either by

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DAPI

FANCD2

MRE11

Merge

MMC (100 ng ml1)

Figure 4 | Co-localization of the NBS1MRE11RAD50 complex with FANCD2 in DNA-damage-inducible subnuclear foci. Telomerase-immortalized NBS fibroblasts were stably transfected with the cDNA encoding wild-type NBS1. The cells were exposed to mitomycin C (MMC) (100 ng/ml), and 24 hours later the cells were examined by immunofluorescence with a polyclonal antibody to FANCD2 (green) and a monoclonal antibody to MRE11 (red). Co-localization of FANCD2 and NBSMRE11RAD50 foci is indicated in the merge panel, in yellow. DAPI staining indicates the nucleus. Adapted from REF. 73 (2002) Macmillan Magazines Ltd.

disrupting the MRE11-binding carboxyl terminus of NBS1 or disrupting the FANCD2 monoubiquitylation site, resulted in mitomycin C hypersensitivity. The MRE11 complex and the FA pathway participate in the cellular response to DNA crosslink formation. In response to ionizing radiation, the NBS1 protein also interacts with ATM to promote phosphorylation of the FANCD2 protein at serine 222. NBS1/ cells are defective in the ATM-dependent phosphorylation of FANCD2. The MRE11 complex and the FA pathway therefore participate in the S-phase checkpoint response. The NBS1 protein functions at the intersection of two signalling pathways and interacts with FANCD2 in each case (FIG. 3). In response to ionizing radiation, ATM phosphorylates NBS1 on serine 343, leading to phosphorylation of FANCD2 and the establishment of an S-phase checkpoint response. In response to mitomycin C, NBS1 assembles in nuclear foci with MRE11/RAD50 and FANCD2 (FIG. 4). Formation of this complex correlates with mitomycin C resistance. The two cellular functions of the NBS1 protein can be dissociated experimentally. First, the ionizing-radiation-inducible phosphorylation of NBS1 on serine 343 is independent of MRE11 binding and foci assembly. A mutant form of NBS1 that lacks the carboxy-terminal MRE11 binding domain does induce the ATM-dependent ionizing-radiation-inducible S-phase checkpoint response, even though it fails to assemble in ionizing-radiation-inducible foci. Second, the ability of NBS1 to bind MRE11 and to co-localize with activated FANCD2 is independent of phosphorylation at serine 343. Accordingly, the phosphorylation-defective mutant of NBS1 (S343A) assembles in DNA-damage-inducible nuclear foci and confers mitomycin C resistance. The precise nature of the interaction between FANCD2 and NBS during the intra-S-phase checkpoint response or the DNA-repair response remains unclear.
Novel diagnostic approaches

post-translational modifications to the FANCD2 protein, by western blotting, can provide insights into a patients genetic defect. Several FA proteins are required for the DNA-damage-induced monoubiquitylation of FANCD2, whereas the ionizing-radiationinducible phosphorylation of FANCD2 requires ATM, NBS and MRE11. FA can also be distinguished in this manner from several other chromosome breakage disorders, including AT, NBS and Bloom syndrome74. Once FA is diagnosed, subtyping of the FA patient is possible, through immunoassays or complementation of cells with retroviruses that encode the FA genes. Identification of the specific FA-inducing mutation is important, as specific mutations have been associated with certain phenotypic consequences. Patients with FA subtypes C and G have more rapid onset of bone-marrow failure, perhaps warranting earlier bone-marrow transplantation. Patients with AT and NBS develop a different spectrum of malignancies and have enhanced radiation sensitivity. FANCD2 monoubiquitylation can also be detected in tumour cells, providing a convenient measure of the integrity of the pathway. Several cancer cell lines particularly ovarian cancer cell lines have defects in FANCD2 monoubiquitylation (A.D.D., unpublished observations). The defects are evident in tumours with a FA-like phenotype (increased chromosome instability and crosslinker hypersensitivity).
Mouse models of Fanconi anaemia

Patients with chromosome instability syndromes have many overlapping clinical features and overlapping cellular phenotypes, so how can FA patients be conclusively identified? A systematic evaluation of the

All human FA-associated genes have mouse homologues. The phenotypes of Fancc 7578, Fanca and Fancg 79,80-null mice are indistinguishable, supporting a model in which the components of the FA nuclear complex cooperate to perform the same function. Furthermore, Fanca/Fancc double-mutants have the same phenotype as single mutants77. The phenotypes of Fanca-, Fancc- and Fancg-null mice will therefore be described together. The phenotype of FA mice is not as severe as that of humans. The lifespan of the animals is normal, and no anaemia or increased tumour incidence has been observed. Haematopoietic cells of FA mice are more susceptible to apoptosis and anaemia, and these mice

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Box 2 | DNA crosslink repair DNA crosslinking agents are cytotoxic not only to FA cells, but also to BRCA1- and BRCA2mutant cells. A close look at the features of mammalian crosslink repair might shed some light on the potential functions of the FA/BRCA proteins. DNA crosslink (dark brown bar) repair is believed to occur through several mechanisms, including intrachromosomal recombination, non-homologous end-joining, homologous recombination or excision/bypass (diagram part a). Although little is known about crosslink repair in mammalian cells, one model based on work from several laboratories has been proposed (diagram part b)117. The model includes components of the nucleotide-excision repair and double-strand-break repair pathways117. One endonuclease complex the XPFERCC1 complex is responsible for incision of the DNA near the crosslink118,119. In addition, double-strand breaks (DSB) are generated near the incised crosslink possibly at the time that the lesion is encountered by replication forks. Because mutations in genes that regulate homologous recombination cause hypersensitivity to crosslinking agents, this process is believed to repair double-strand breaks120. Recent evidence implicates the FA/BRCA pathway in this homologous recombination phase of this repair process. Panel a shows a hypothetical mechanism for the repair of crosslinks that are incurred prior to DNA replication. Only recombination with the homologous chromosome (purple box) would be error-free (non-mutagenic). Yellow boxes indicate mutagenic repair pathways. Panel b shows a model of crosslink repair (modified from REF. 117). Although the proposed model only invokes one double-strand break near the crosslink, the structure of a processed crosslink lesion is not known and could contain up to four double-strand breaks. The FA/BRCA pathway might work at one or more levels in this DNA-repair response.
a
Intrachromosomal recombination Non-homologous end-joining

Stalled replication fork

Unhooking and formation of DSB

2 DSB on same side

2 DSB flanking ICL

Gap resection and recombination

3 DSB

Resolution of recombination

Deletion Homologous recombination ICL

Deletion Excision/bypass

Second excision resynthesis

Misincorporation

HYPOMORPHIC MUTATION

A hypomorphic mutation in a gene partially disrupts the function of the gene. The hypomorphic allele of the gene encodes an abnormal protein with partial cellular function.

have low bodyweight. Subtle haematological defects can be detected by in vitro culture of haematopoietic progenitors76,81. Hypersensitivity to cytokines particularly interferon- has been described82,83, which could underlie the inability of these stem cells to fully repopulate the myeloid and lymphoid lineage84. However, this phenotype has not been observed in all strains of mice77. Cells from all FA mutant mice, conversely, display in vivo and in vitro sensitivity to DNA crosslinking agents8587 and to ionizing radiation86. The FA complex therefore seems to be involved in the cellular responses to DNA-damaging agents in mice as well as humans. Another intriguing phenotype of FA mice is a reduction in the number of germ cells in both males and females.

Fancd2 mutants. The phenotype of Fancd2-null mice has not been published, but these mice seem to have defects that Fancc, Fanca and Fancg mice do not (S. Houghtaling and M. G., unpublished observations). These mice have germ-cell deficiency, are smaller than littermates, and, importantly, have a high incidence of epithelial tumours, starting at age 15 months. Overall, their phenotype is more similar to that of mice with mild Brca2 mutations51,8890. Unlike mice with disruptions in FA-associated genes, however, Brca2-null mice die during embryogenesis90, as do Rad51-null mice91 and Brca1-null mice92,93. Mice with tissue-specific conditional knockouts89 or HYPO51 MORPHIC MUTATIONS have been developed to study these pathways. Mice with disruptions in Brca2, specifically

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in breast epithelial cells, develop adenocarcinoma at a high frequency late in life89, and mutations that cause a carboxy-terminal truncation of Brca2 cause tumours in a variety of tissues51. Therefore, Brca2 gene mutations cause phenotypes that are distinct from those of Fanca, Fancc and Fancg, but are similar to those of Fancd2-mutant mice.
DNA repair in Fanconi anaemia

The precise role of the FA pathway in the DNA crosslinking response is not clear1. DNA crosslink repair involves several steps; the FA pathway could be involved in any of these (BOX 2). After DNA crosslinking occurs, FA cells arrest during late S phase with a SUB-4N DNA CONTENT. The duration of the arrest is 310 times longer in FA cells compared with normal control cells9. The FA proteins might therefore function in an S-phase-specific DNA-damage response9. Consistent with this idea, FA pathway activation, as detected by monoubiquitylation of the FANCD2 protein, occurs specifically during the S phase of the cell cycle46. Several models for the role of FA proteins in S phase have been put forth and will be considered separately. Defects in the S-phase checkpoint. Several studies have described a defect in radiation-induced arrest of DNA synthesis in FA cells94. After exposure to ionizing radiation, wild-type cells undergo cell-cycle arrest within 1545 minutes of DNA synthesis. However, immortalized FA lymphoblasts57 and SV40 T-antigen transformed fibroblasts65 do not arrest at this normal intra-S-phase checkpoint. In the case of FANCD2-mutant cells, radiation-induced arrest of DNA synthesis can be linked to deficiencies in phosphorylation of FANCD2. In addition to the intra-S-phase checkpoint defect, two reports have shown a failure of FA lymphoblasts to arrest DNA synthesis after crosslinking has occurred95,96. The failure of FA cells to block DNA synthesis in response to damage therefore seems to be one reason for their hypersensitivity to DNA-damaging agents. However, wild-type cells also undergo a prolonged cellcycle arrest in late S phase after DNA damage97, despite their ability to stop DNA synthesis after damage. The delay in S-phase progression that is associated with the radiation-induced arrest of DNA synthesis is therefore insufficient to allow time for complete repair of crosslinks. Furthermore, the DNA content of FA cells and normal cells that have undergone S-phase arrest is indistinguishable both are 4N. The FA proteins therefore do not function in a checkpoint pathway that induces early- or mid-S-phase arrest. It is possible that the intact FA pathway temporarily stops DNA synthesis after damage, but it seems unlikely that this is its sole role in the DNA-crosslinking damage response. Defective non-homologous end-joining. The most straightforward mechanism for repair of interstrand crosslinks would entail a simple cut and paste approach. After creation of two double-strand breaks, the chromatids could be rejoined on each side of the interstrand crosslink, leaving a small deletion. Two

groups have reported that FA cells have end-joining defects. The ability of nuclear extracts from both FA cells and wild-type cells to rejoin cleaved plasmid DNA ends were tested in vitro98,99. FA cell extracts functioned abnormally in this assay, as plasmids that were treated with these extracts had more numerous and larger deletions99,100. These results indicate that FA cells are defective in their level of DNA end-joining activity. It is difficult, however, to interpret the relevance of these in vitro end-joining experiments because the concentration of substrate (plasmid DNA ends) was much higher than the expected physiological amounts of lesions in an intact cell53. Defective homologous recombination. The recent discovery that BRCA2 is mutated in FA complementation groups D1 and B indicates that the FA pathway might function in homologous recombination during S phase. Several studies have implicated BRCA2 in DNA repair by homologous recombination101104, and have shown that BRCA2-null cells are defective in homologous recombination102,103. BRCA2 directly binds the DNArepair protein RAD51 (REF. 105), and increases levels of RAD51-mediated recombination in vitro106,107. So BRCA2 might function to enhance RAD51 function and promote homologous recombination during S phase101. But how does this fit in with the FA pathway? The subcellular localization of monoubiquitylated FANCD2 is consistent with a role in mediating DNA recombination. During meiosis of male cells, Fancd2 co-localizes with Brca1 (REFS 45,108). Antibodies against Fancd2 label meiotic chromosomes at synaptonemal complexes45, which represent sites of meiotic recombination109. Defects of homologous recombination for DNA repair have been found in both Brca1- and Brca2mutant cells110,111, as well as in pachytene sperm from Fanca- and Fancc-null mice112. Not many studies have directly analysed homologous recombination in FA cells, however. One compared the ability of nuclear extracts from normal and FA cells to induce homologous recombination of plasmid substrates, and the FA cells were found to have increased homologous recombination activity113. The recombination substrate used did not, however, distinguish error-free from error-prone recombination, so it is still possible that errors occur during this process in FA cells103. The increased recombination activity in FA cells could simply reflect the presence of unrepaired double-strand breaks or other defects that occur during DNA recombination. Few studies have evaluated the mutation frequency of FA cells. When the HPGRT locus was analysed as a mutation target, FA cells were found to show an abnormal spectrum of mutations, with a notable increase in deletions 114116. These observations are again consistent with the idea that FA cells are defective in a non-mutagenic repair pathway, such as error-free homologous recombination. Finally, FA- and BRCA-mutant cells show quadraradial chromosomes after exposure to genotoxic stresses. The precise molecular nature of chromosome

SUB-4N DNA CONTENT

A cell with sub-4N DNA content has not completed full DNA replication. By definition, the cell is undergoing DNA replication.

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radials is not known, but they seem to represent abnormal conjoining of non-homologous autosomes at metaphase. The presence of chromosome radials could signify aberrant recombination between nonhomologous or only partially homologous chromosomes. In this view, radials could be the consequence of a general hyper-recombination phenotype, leading to mis-recombination. The FA/BRCA pathway might therefore function to provide fidelity in the homologous recombination process.
Other possibilities

It is therefore likely that the FA pathway only regulates the rate of homologous recombination or the nature of homologous recombination events.
Future directions

If the FA proteins function with BRCA2 in an S-phase-specific homologous-recombination-repair pathway, it will be important to determine how recombination events are controlled by this pathway. Several different possibilities can be considered. For example, the FA complex could be involved in sensing DNA damage and recruiting and organizing repair proteins such as BRCA2/RAD51. It could also be involved in processing a lesion that blocks replication, such as by inducing the formation of double-strand breaks. The FA complex might also function to assess the quality of homologous recombination and prevent recombination with non-homologous sequences, or it could scan the genome for non-homologous chromosome sequences. Whatever the role of the FA pathway, its function is not absolutely required for homologous recombination. Unlike Rad51- null 91 Brca2- null 90 animals, FA-defective animals and humans can complete embryonic development and live for many years before significant manifestations of the disease occur.

Despite recent progress in our understanding of FA pathogenesis, several questions remain. First, it will be important to identify other FA genes and their encoded proteins. Recent studies indicate the existence of more FA complementation groups. Additional FA genes could be required to maintain normal cellular resistance to genotoxic agents. Furthermore, the relationship between the FA/BRCA pathway and homologous-recombination DNA repair remains obscure. It will be important to clarify this interaction, and to determine the precise function of FANCD2 monoubiquitylation in the regulation of the downstream DNA-repair events. Preliminary studies indicate that this is a crucial targeting event that allows the FANCD2 protein to accumulate at sites of damaged chromatin. Finally, sporadic tumours can be screened for acquired disruptions of the FA pathway. Detection of FA mutations in tumour samples could be of prognostic or therapeutic value. For example, a tumour that is caused by defects in the FA pathway could be more sensitive to crosslinking agents than to ionizing-radiation therapy. Improving our understanding of the molecular mechanisms of the FA pathway might also lead to the design of therapeutic agents to decrease FA-protein function, perhaps by altering levels of FANCD2 monoubiquitylation in tumour cells to make them more sensitive to chemotherapy.

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Acknowledgements
We apologize to those authors whose work is not cited owing to space constraints. A.D.D. is a Doris Duke Distinguished Clinical Scientist and is supported by National Institutes of Health grants. M.G. is supported by a National Institutes of Health grant.

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Online links
DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ ATM | BRCA1 | BRCA2 | FANCA | FANCC | FANCD1 | FANCD2 | FANCE | FANCF | FANCG | HPGRT | MRE11 | NBS1 | RAD6 | RAD18 | RAD51 OMIM: http://www.ncbi.nlm.nih.gov/Omim/ ataxia telangiectasia | Bloom syndrome | Fanconi anaemia | Nijmegen breakage syndrome Access to this interactive links box is free online.

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