A ubiquitin-binding protein, FAAP20, links RNF8-mediated ubiquitination to the Fanconi anemia DNA repair network

The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.     

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.     

New Advances in the DNA Damage Response Network of Fanconi Anemia and BRCA proteins: FAAP95 Replaces BRCA2 as the True FANCB Protein

Fanconi anemia (FA) proteins function in a DNA damage response pathway that appears to be part of the network including breast cancer susceptibility gene products, BRCA1 and BRCA2. In response to DNA damage or replication signals, a nuclear FA core complex of at least 6 FA proteins (FANCA, FANCC, FANCE, FANCF, FANCG, and FANCL) is activated and leads to monoubiquitination of the downstream FA protein, FANCD2. One puzzling question for this pathway is the role of BRCA2. A previous study has proposed that BRCA2 could be identical to two FA proteins: FANCD1, which functions either downstream or in a parallel pathway; and FANCB, which functions upstream of the FANCD2 monoubiquitination. Now, a new study shows that the real FANCB protein is not BRCA2, but a previously uncharacterized component of the FA core complex, FAAP95, suggesting that BRCA2 does not act upstream of the FA pathway. Interestingly, the newly discovered FANCB gene is X-linked and subject to X-inactivation. The presence of a single active copy of FANCB and its essentiality for a functional FA-BRCA pathway make it a potentially vulnerable component of the cellular machinery that maintains genomic integrity.     

Monoubiquitination-dependent chromatin loading of FancD2 in silkworms, a species lacking the FA core complex

The Fanconi anemia (FA) pathway is required for activation and operation of the DNA interstrand cross-link (ICL) repair pathway, although the precise mechanism of the FA pathway remains largely unknown. A critical step in the FA pathway is the monoubiquitination of FANCD2 catalyzed by a FA core complex. This modification appears to allow FANCD2 to coordinate ICL repair with other DNA repair proteins on chromatin. Silkworm, Bombyx mori, lacks apparent homologues of the FA core complex. However, BmFancD2 and BmFancI, the putative substrates of the complex, and BmFancL, the putative catalytic E3 ubiquitin ligase, are conserved. Here, we report that the silkworm FancD2 is monoubiquitinated depending on FancI and FancL, and stabilized on chromatin, following MMC treatment. A substitution of BmFancD2 at lysine 519 to arginine abolishes the monoubiquitination, but not the interaction between the FancD2 and FancI. In addition, we demonstrated that depletion of BmFancD2, BmFancI or BmFancL had effects on cell proliferation in the presence of MMC. These results suggest that the FA pathway in B. mori works in the same manner as that in vertebrates.     

Dedicated to the core: understanding the Fanconi anemia complex

The Fanconi anemia (FA) pathway consists of a unique, multi-subunit E3 ubiquitin ligase complex that is activated in a replication and DNA-damage dependent mechanism. This FA core complex possesses a putative helicase and an E3 ubiquitin ligase subunit, is assembled in both the nucleoplasm and in chromatin, and is required for the mono-ubiquitination of FANCD2, a downstream FA protein, following genotoxic stress. Clinically, absence of the FA pathway results in congenital defects, bone marrow failure, and cancer predisposition. At the cellular level, this pathway is required for chromosomal stability and cellular resistance to DNA interstrand crosslinkers (ICLs) such as mitomycin C (MMC). A general model has emerged for the FA pathway as an arm of the DNA-damage response following ICLs. This review will summarize the current understanding of the FA core complex and propose a model for its activity.     

FANCD2, FANCJ and BRCA2 cooperate to promote replication fork recovery independently of the Fanconi Anemia core complex

Fanconi Anemia (FA) is an inherited multi-gene cancer predisposition syndrome that is characterized on the cellular level by a hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA pathway proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex monoubiquitinates the central FA pathway members FANCD2 and FANCI, followed by their recruitment to chromatin. Chromatin-bound monoubiquitinated FANCD2 and FANCI subsequently coordinate DNA repair factors including the downstream FA pathway members FANCJ and FANCD1/BRCA2 to repair the DNA ICL. Importantly, we recently showed that FANCD2 has additional independent roles: it binds chromatin and acts in concert with the BLM helicase complex to promote the restart of aphidicolin (APH)-stalled replication forks, while suppressing the firing of new replication origins. Here, we show that FANCD2 fulfills these roles independently of the FA core complex-mediated monoubiquitination step. Following APH treatment, nonubiquitinated FANCD2 accumulates on chromatin, recruits the BLM complex, and promotes robust replication fork recovery regardless of the absence or presence of a functional FA core complex. In contrast, the downstream FA pathway members FANCJ and BRCA2 share FANCD2''s role in replication fork restart and the suppression of new origin firing. Our results support a non-linear FA pathway model at stalled replication forks, where the nonubiquitinated FANCD2 isoform – in concert with FANCJ and BRCA2 – fulfills a specific function in promoting efficient replication fork recovery independently of the FA core complex.     

FANCD2, FANCJ and BRCA2 cooperate to promote replication fork recovery independently of the Fanconi Anemia core complex

Fanconi Anemia (FA) is an inherited multi-gene cancer predisposition syndrome that is characterized on the cellular level by a hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA pathway proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex monoubiquitinates the central FA pathway members FANCD2 and FANCI, followed by their recruitment to chromatin. Chromatin-bound monoubiquitinated FANCD2 and FANCI subsequently coordinate DNA repair factors including the downstream FA pathway members FANCJ and FANCD1/BRCA2 to repair the DNA ICL. Importantly, we recently showed that FANCD2 has additional independent roles: it binds chromatin and acts in concert with the BLM helicase complex to promote the restart of aphidicolin (APH)-stalled replication forks, while suppressing the firing of new replication origins. Here, we show that FANCD2 fulfills these roles independently of the FA core complex-mediated monoubiquitination step. Following APH treatment, nonubiquitinated FANCD2 accumulates on chromatin, recruits the BLM complex, and promotes robust replication fork recovery regardless of the absence or presence of a functional FA core complex. In contrast, the downstream FA pathway members FANCJ and BRCA2 share FANCD2's role in replication fork restart and the suppression of new origin firing. Our results support a non-linear FA pathway model at stalled replication forks, where the nonubiquitinated FANCD2 isoform – in concert with FANCJ and BRCA2 – fulfills a specific function in promoting efficient replication fork recovery independently of the FA core complex.     

The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway

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

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

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

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.     

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.     

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.     

Ubiquitylation and the Fanconi anemia pathway

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability.     

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.     

Positional cloning of a novel Fanconi anemia gene, FANCD2

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.     

范可尼贫血与泛素化

Fanconi贫血是一种罕见的隐性遗传性疾病,临床常以先天性畸形、进行性骨髓衰竭和遗传性肿瘤倾向为主要表现而确诊。FA病人细胞对DNA交联剂如丝裂霉素C (MMC)高度敏感。目前已经发现至少12种FA基因的缺失或突变能够引起FA表型的出现,其中10种相应的编码蛋白形成FA复合物共同参与FA/BRCA2 DNA损伤修复途径—FA途径。FA核心复合物蛋白FANCL具有泛素连接酶活性,在结合酶UBE2T共同作用下,催化下游蛋白FANCD2单泛化,泛素化FANCD2与BRCA2形成新的复合物,修复DNA损伤。去泛素化酶USP1在DNA修复完毕后移除FANCD2的单体泛素,使因损伤修复而阻滞的细胞周期继续进行。机体很可能在不同信号通路对FANCD2泛素化/去泛素化的精细调节下,调控FA途径参与不同的DNA修复过程。     

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.     

Mutations in the Gene Encoding the E2 Conjugating Enzyme UBE2T Cause Fanconi Anemia

Fanconi anemia (FA) is a rare genetic disorder characterized by genome instability, increased cancer susceptibility, progressive bone marrow failure (BMF), and various developmental abnormalities resulting from the defective FA pathway. FA is caused by mutations in genes that mediate repair processes of interstrand crosslinks and/or DNA adducts generated by endogenous aldehydes. The UBE2T E2 ubiquitin conjugating enzyme acts in FANCD2/FANCI monoubiquitination, a critical event in the pathway. Here we identified two unrelated FA-affected individuals, each harboring biallelic mutations in UBE2T. They both produced a defective UBE2T protein with the same missense alteration (p.Gln2Glu) that abolished FANCD2 monoubiquitination and interaction with FANCL. We suggest this FA complementation group be named FA-T.     

New Roads to FA/BRCA Pathway: H2AX

We have recently described an involvement of H2AX into the Fanconi anemia (FA) BRCA pathway through recruitment of FA protein FANCD2 to the sites of stalled replication forks. We showed that BRCA1 mediates the recruitment of FANCD2 by γH2AX to damaged chromatin and cells deficient or depleted of H2AX exhibit an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. Here, we discuss a model for the FA pathway and how it could partially explain the common phenotypes of H2AX, BRCA2 and FA deficiencies.     

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.