FANCI Protein Binds to DNA and Interacts with FANCD2 to Recognize Branched Structures

In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200–1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001–1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.Fanconi anemia (FA)³ is a genetic disorder characterized by chromosome instability, predisposition to cancer, hypersensitivity to DNA cross-linking agents, developmental abnormalities, and bone marrow failure (1–9). There are at least 13 distinct FA complementation groups, each of which is associated with an identified gene (2, 9, 10). Eight of them are components of the FA core complex (FANC A, B, C, E, F, G, L, and M) that is epistatic to the monoubiquitination of both FANCI and FANCD2, a key event to initiate interstrand cross-link (ICL) repair (2, 9, 11). Downstream of or parallel to the FANCI and FANCD2 monoubiquitination are the proteins involved in double strand break repair and breast cancer susceptibility (i.e. FANCD1/BRCA2, FANCJ/BRIP1, and FANCN/PALB2) (2, 9).FANCI is the most recently identified FA gene (11–13). FANCI protein is believed to form a FANCI-FANCD2 (ID) complex with FANCD2, because they co-immunoprecipitate with each other from cell lysates and their stabilities are interdependent of each other (9, 11, 13). FANCI and FANCD2 are paralogs to each other, since they share sequence homology and co-evolve in the same species (11). Both FANCI and FANCD2 can be phosphorylated by ATR/ATM (ataxia telangiectasia and Rad3-related/ataxia telangiectasia-mutated) kinases under genotoxic stress (11, 14, 15). The phosphorylation of FANCI seems to function as a molecular switch to turn on the FA repair pathway (16). The monoubiquitination of FANCD2 at lysine 561 plays a critical role in cellular resistance to DNA cross-linking agents and is required for FANCD2 to form damage-induced foci with BRCA1, BRCA2, RAD51, FANCJ, FANCN, and γ-H2AX on chromatin during S phase of the cell cycle (17–25). In response to DNA damage or replication stress, FANCI is also monoubiquitinated at lysine 523 and recruited to the DNA repair nuclear foci (11, 13). The monoubiquitination of both FANCI and FANCD2 depends on the FA core complex (11, 13, 26), and the ubiquitination of FANCI relies on the FANCD2 monoubiquitination (2, 11). In an in vitro minimally reconstituted system, FANCI enhances FANCD2 monoubiquitination and increases its specificity toward the in vivo ubiquitination site (27).FANCI is a leucine-rich peptide (14.8% of leucine residues) with limited sequence information to indicate which processes it might be involved in. Besides the monoubiquitination site Lys⁵²³ and the putative nuclear localization signals (Fig. 1A), FANCI contains both ARM (armadillo) repeats and a conserved C-terminal EDGE motif as FANCD2 does (11, 28). The EDGE sequence in FANCD2 is not required for monoubiquitination but is required for mitomycin C (MMC) sensitivity (28). The ARM repeats form α-α superhelix folds and are involved in mediating protein-protein interactions (11, 29). In addition, FANCI, at its N terminus, contains a leucine zipper domain (aa 130–151) that could be involved in mediating protein-protein or protein-DNA interactions (Fig. 1A) (30–33). FANCD2, the paralog of FANCI, was reported to bind to double strand DNA ends and Holliday junctions (34).Open in a separate windowFIGURE 1.Purified human FANCI binds to DNA promiscuously. A, schematic diagram of predicted FANCI motifs and mutagenesis strategy to define the DNA binding domain. The ranges of numbers indicate how FANCI was truncated (e.g. 801–1328 represents FANCI-(801–1328)). NLS, predicted nuclear localization signal (aa 779–795 and 1323–1328); K523, lysine 523, the monoubiquitination site. The leucine zipper (orange bars, aa 130–151), ARM repeats (green bars), and EDGE motif (blue bars) are indicated. Red bars with a slash indicate the point mutations shown on the left. B, SDS-PAGE of the purified proteins stained with Coomassie Brilliant Blue R-250. R1285Q and D1301A are two point mutants of FANCI. All FANCI variants are tagged by hexahistidine. FANCD2 is in its native form. Protein markers in kilodaltons are indicated. C, titration of WT-FANCI for the DNA binding activity. Diagrams of the DNA substrates are shown at the top of each set of reactions. *, ³²P-labeled 5′-end. HJ, Holliday junction. Concentrations of FANCI were 0, 20, 40, 60, and 80 nm (ascending triangles). The substrate concentration was 1 nm. Protein-DNA complex is indicated by an arrow. D, supershift assay. 1 nm of ssDNA was incubated with PBS (lane 1), 80 nm FANCI alone (lane 2), and 80 nm FANCI preincubated with a specific FANCI antibody (lane 3) in the condition described under “Experimental Procedures.”In order to delineate the function of FANCI protein, we purified the recombinant FANCI from the baculovirus expression system. In this study, we report the DNA binding activity of FANCI. Unlike FANCD2, FANCI binds to different DNA structures, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), 5′-tailed, 3′-tailed, splayed arm, 5′-flap, 3′-flap, static fork, and Holliday junction with preference toward branched structures in the presence of FANCD2. Our data suggest that the dynamic DNA binding activity of FANCI and the preferential recognition of branched structures by the ID complex are likely to be the mechanisms to initiate downstream repair events.     

FANCD2 regulates BLM complex functions independently of FANCI to promote replication fork recovery

Fanconi Anemia (FA) and Bloom Syndrome share overlapping phenotypes including spontaneous chromosomal abnormalities and increased cancer predisposition. The FA protein pathway comprises an upstream core complex that mediates recruitment of two central players, FANCD2 and FANCI, to sites of stalled replication forks. Successful fork recovery depends on the Bloom’s helicase BLM that participates in a larger protein complex (‘BLMcx’) containing topoisomerase III alpha, RMI1, RMI2 and replication protein A. We show that FANCD2 is an essential regulator of BLMcx functions: it maintains BLM protein stability and is crucial for complete BLMcx assembly; moreover, it recruits BLMcx to replicating chromatin during normal S-phase and mediates phosphorylation of BLMcx members in response to DNA damage. During replication stress, FANCD2 and BLM cooperate to promote restart of stalled replication forks while suppressing firing of new replication origins. In contrast, FANCI is dispensable for FANCD2-dependent BLMcx regulation, demonstrating functional separation of FANCD2 from FANCI.     

Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair

Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.     

Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2-FANCI complex versus the monomeric proteins are. We show that the FANCD2-FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2-FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to-and independently of-FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase.     

Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.     

FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2

The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.     

Defective FANCI Binding by a Fanconi Anemia-Related FANCD2 Mutant

FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2^−/− DT40 cells. Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation.     

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.     

FANCI Binds Branched DNA and Is Monoubiquitinated by UBE2T-FANCL

FANCI is integral to the Fanconi anemia (FA) pathway of DNA damage repair. Upon the occurrence of DNA damage, FANCI becomes monoubiquitinated on Lys-523 and relocalizes to chromatin, where it functions with monoubiquitinated FANCD2 to facilitate DNA repair. We show that FANCI and its C-terminal fragment possess a DNA binding activity that prefers branched structures. We also demonstrate that FANCI can be ubiquitinated on Lys-523 by the UBE2T-FANCL pair in vitro. These findings should facilitate future efforts directed at elucidating molecular aspects of the FA pathway.Fanconi anemia (FA)⁴ is characterized by developmental defects, bone marrow failure, and a strong predisposition to cancer. FA cells exhibit exquisite sensitivity to DNA cross-linking agents and marked genomic instability, indicative of a failure to repair damaged DNA (1–3). Thirteen FA proteins have been identified, of which eight, FANC-A, -B, -C, -E, -F, -G, -L, and -M, form part of a nuclear core complex that is required to monoubiquitinate two other FA proteins, FANCD2 and FANCI. When monoubiquitinated, FANCD2 and FANCI become chromatin-associated in foci that contain various factors, including the RAD51 recombinase BRCA2 (also known as FANCD1) and PALB2 (also called FANCN), which mediate DNA repair via RAD51-catalyzed homologous recombination (4).Monoubiquitination of FANCD2 appears to be a key event for proper repair of exogenous DNA damage but also occurs during an unperturbed S phase, likely in response to stalled replication forks (4–7). FANCD2 monoubiquitination depends on the E3 ligase activity of FANCL (8) and on the E2 ubiquitin-conjugating enzyme, UBE2T (9). In vitro, FANCL and UBE2T can monoubiquitinate chicken FANCD2 (10).FANCI was identified recently as a target protein for the ATM/ATR kinase. FANCI is also monoubiquitinated, in a manner that is dependent on the FA core complex (11). In cells, a fraction of FANCD2 and FANCI associates in a complex. Moreover, the amount and monoubiquitination of these two FA proteins are co-dependent in human cells, i.e. the quantity and monoubiquitination of FANCD2 are diminished in FANCI-deficient cells and vice versa (11–14). These observations suggest that FANCI and FANCD2 form a complex integral to cellular DNA repair capacity. Mutating the ubiquitinated target lysine of FANCI (Lys-523) renders cells sensitive to DNA damage and impairs the assembly of DNA damage-induced nuclear foci of FANCD2 and FANCI (11, 14). Herein, we document studies that reveal several biochemical attributes of FANCI, including DNA binding, and its monoubiquitination, that are relevant for understanding the biological role of this key FA protein.     

Interaction of FANCD2 and NBS1 in the DNA damage response

Fanconi anaemia (FA) and Nijmegen breakage syndrome (NBS) are autosomal recessive chromosome instability syndromes with distinct clinical phenotypes. Cells from individuals affected with FA are hypersensitive to mitomycin C (MMC), and cells from those with NBS are hypersensitive to ionizing radiation. Here we report that both NBS cell lines and individuals with NBS are hypersensitive to MMC, indicating that there may be functional linkage between FA and NBS. In wild-type cells, MMC activates the colocalization of the FA subtype D2 protein (FANCD2) and NBS1 protein in subnuclear foci. Ionizing radiation activates the ataxia telangiectasia kinase (ATM)-dependent and NBS1-dependent phosphorylation of FANCD2, resulting in an S-phase checkpoint. NBS1 and FANCD2 therefore cooperate in two distinct cellular functions, one involved in the DNA crosslink response and one involved in the S-phase checkpoint response.     

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.     

Drosophila homologs of FANCD2 and FANCL function in DNA repair

Fanconi anemia (FA) is a genetically heterogeneous disease characterized by developmental defects, progressive bone marrow failure and cancer susceptibility. Cells derived from patients with FA show spontaneous chromosomal aberrations and hypersensitivity to cross-linking agents, indicating a cellular defect in DNA repair. Among the 12 FA genes, only FANCD2, FANCL and FANCM have Drosophila homologs. Given this difference between the human and Drosophila FA pathways, it is unknown whether the fly homologs function in DNA repair. Here, we report that knockdown of Drosophila FANCD2 or FANCL leads to specific hypersensitivity to cross-linking agents. Further analysis revealed that FANCD2 and FANCL function in a linear pathway with FANCL being necessary for the monoubiquitination of FANCD2. FANCD2 mutants also exhibited the same defect in the ionizing radiation-inducible S-phase checkpoint that is seen in mammalian cells deficient for this gene. Finally, in an assay for inactivating mutations, FANCD2 mutants have an elevated mutation rate in response to nitrogen mustard, indicating that these flies are hypermutable. Taken together, these data demonstrate that Drosophila FANCD2 and FANCL play a critical role in DNA repair. Because of the lack of other FA genes, further studies will determine whether the conserved FA genes function as the minimal machinery or whether additional genes are involved in the Drosophila FA pathway.     

Deubiquitination of FANCD2 is required for DNA crosslink repair

Monoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.     

AHNAK interacts with the DNA ligase IV-XRCC4 complex and stimulates DNA ligase IV-mediated double-stranded ligation

AHNAK is a high molecular weight protein that is under-expressed in several radiosensitive neuroblastoma cell lines. Using immunoaffinity purification or purified proteins, we show that AHNAK interacts specifically with the DNA ligase IV-XRCC4 complex, a complex that functions in DNA non-homologous end-joining. Furthermore, AHNAK and the DNA ligase IV-XRCC4 complex co-immunoprecipitate demonstrating an in vivo interaction. This interaction is specific and is not observed with other DNA ligases nor with other components of the DNA non-homologous end-joining machinery. We characterised AHNAK as a protein that stimulates the double-stranded (DS) ligation activity of DNA ligase IV-XRCC4. We show that AHNAK has weak DNA-binding activity and forms a stable complex with the DNA ligase IV-XRCC4 complex on DNA. AHNAK is also able to link two DNA molecules to a similar extent to that previously reported for Ku. Together, these findings demonstrate new activities for AHNAK, and raise the possibility that it may function to modulate DNA non-homologous end-joining.     

UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination

The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.     

Functional interaction of monoubiquitinated FANCD2 and BRCA2/FANCD1 in chromatin

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

DNA structure-induced recruitment and activation of the Fanconi anemia pathway protein FANCD2

The Fanconi anemia (FA) pathway proteins are thought to be involved in the repair of irregular DNA structures including those encountered by the moving replication fork. However, the nature of the DNA structures that recruit and activate the FA proteins is not known. Because FA proteins function within an extended network of proteins, some of which are still unknown, we recently established cell-free assays in Xenopus laevis egg extracts to deconstruct the FA pathway in a fully replication-competent context. Here we show that the central FA pathway protein, xFANCD2, is monoubiquitinated (xFANCD2-L) rapidly in the presence of linear and branched double-stranded DNA (dsDNA) structures but not single-stranded or Y-shaped DNA. xFANCD2-L associates with dsDNA structures in an FA core complex-dependent manner but independently of xATRIP, the regulatory subunit of xATR. Formation of xFANCD2-L is also triggered in response to circular dsDNA, suggesting that dsDNA ends are not required to trigger monoubiquitination of FANCD2. The induction of xFANCD2-L in response to circular dsDNA is replication and checkpoint independent. Our results provide new evidence that the FA pathway discriminates among DNA structures and demonstrate that triggering the FA pathway can be uncoupled from DNA replication and ATRIP-dependent activation.     

MEN1 and FANCD2 mediate distinct mechanisms of DNA crosslink repair

Cells mutant for multiple endocrine neoplasia type I (MEN1) or any of the Fanconi anemia (FA) genes are hypersensitive to the killing effects of crosslinking agents, but the precise roles of these genes in the response to interstrand crosslinks (ICLs) are unknown. To determine if MEN1 and the FA genes function cooperatively in the same repair process or in distinct repair processes, we exploited Drosophila genetics to compare the mutation frequency and spectra of MEN1 and FANCD2 mutants and to perform genetic interaction studies. We created a novel in vivo reporter system in Drosophila based on the supF gene and showed that MEN1 mutant flies were extremely prone to single base deletions within a homopolymeric tract. FANCD2 mutants, on the other hand, had a mutation frequency and spectrum similar to wild type using this assay. In contrast to the supF results, both MEN1 and FANCD2 mutants were hypermutable using a different assay based on the lats tumor suppressor gene. The lats assay showed that FANCD2 mutants had a high frequency of large deletions, which the supF assay was not able to detect, while large deletions were rare in MEN1 mutants. Genetic interaction studies showed that neither overexpression nor loss of MEN1 modified the ICL sensitivity of FANCD2 mutants. The strikingly different mutation spectra of MEN1 and FANCD2 mutants together with lack of evidence for genetic interaction between these genes indicate MEN1 plays an essential role in ICL repair distinct from the Fanconi anemia genes.     

FANCD2 is a target for caspase 3 during DNA damage-induced apoptosis

The Fanconi anemia (FA) pathway, of which the FANCD2 protein is a key component, plays crucial roles in the maintenance of hematopoietic stem cells and suppression of carcinogenesis. However, the function of FANCD2 remains unclear. Here, we report that FANCD2 is a novel and specific substrate of caspase 3. Cleavage of FANCD2 by caspase 3 did not require either the FA core complex or mono-ubiquitylation of FANCD2, and was stimulated by p53. In addition, we identified the cleavage sites and generated cell lines that stably express a caspase-resistant FANCD2 mutant. Our data suggest that FANCD2 is regulated by caspase-mediated degradation during apoptosis induced by DNA damage.     

FANCC, FANCE, and FANCD2 form a ternary complex essential to the integrity of the Fanconi anemia DNA damage response pathway

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, cancer predisposition, and increased cellular sensitivity to DNA-cross-linking agents. The products of seven of the nine identified FA genes participate in a protein complex required for monoubiquitination of the FANCD2 protein. Direct interaction of the FANCE protein with both fellow FA complex component FANCC and the downstream FANCD2 protein has been observed in the yeast two-hybrid system. Here, we demonstrate the ability of FANCE to mediate the interaction between FANCC and FANCD2 in the yeast three-hybrid system and confirm the FANCE-mediated association of FANCC with FANCD2 in human cells. A yeast two-hybrid system-based screen was devised to identify randomly mutagenized FANCE proteins capable of interaction with FANCC but not with FANCD2. Exogenous expression of these mutants in an FA-E cell line and subsequent evaluation of FANCD2 monoubiquitination and DNA cross-linker sensitivity indicated a critical role for the FANCE/FANCD2 interaction in maintaining FA pathway integrity. Three-hybrid experiments also demonstrated the ability of FANCE to mediate the interaction between FA core complex components FANCC and FANCF, indicating an additional role for FANCE in complex assembly. Thus, FANCE is shown to be a key mediator of protein interactions both in the architecture of the FA protein complex and in the connection of complex components to the putative downstream targets of complex activity.