UBE2W interacts with FANCL and regulates the monoubiquitination of fanconi anemia protein FANCD2

Fanconi anemia (FA) is a rare cancer-predisposing genetic disease mostly caused by improper regulation of the monoubiquitination of Fanconi anemia complementation group D2 (FANCD2). Genetic studies have indicated that ubiquitin conjugating enzyme UBE2T and HHR6 could regulate FANCD2 monoubiquitination through distinct mechanisms. However, the exact regulation mechanisms of FANCD2 monoubiquitination in response to different DNA damages remain unclear. Here we report that UBE2W, a new ubiquitin conjugating enzyme, could regulate FANCD2 monoubiquitination by mechanisms different from UBE2T or HHR6. Indeed, UBE2W exhibits ubiquitin conjugating enzyme activity and catalyzes the monoubiquitination of PHD domain of Fanconi anemia complementation group L (FANCL) in vitro. UBE2W binds to FANCL, and the PHD domain is both necessary and sufficient for this interaction in mammalian cells. In addition, over-expression of UBE2W in cells promotes the monoubiquitination of FANCD2 and down-regulated UBE2W markedly reduces the UV irradiation-induced but not MMC-induced FANCD2 monoubiquitination. These results indicate that UBE2W regulates FANCD2 monoubiquitination by mechanisms different from UBE2T and HRR6. It may provide an additional regulatory step in the activation of the FA pathway.     

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.     

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.     

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。     

RAD18-mediated ubiquitination of PCNA activates the Fanconi anemia DNA repair network

The Fanconi anemia (FA) network is important for the repair of interstrand DNA cross-links. A key event in FA pathway activation is the monoubiquitylation of the FA complementation group I (FANCI)-FANCD2 (ID) complex by FA complementation group L (FANCL), an E3 ubiquitin ligase. In this study, we show that RAD18, another DNA damage-activated E3 ubiquitin ligase, also participates in ID complex activation by ubiquitylating proliferating cell nuclear antigen (PCNA) on Lys164, an event required for the recruitment of FANCL to chromatin. We also found that monoubiquitylated PCNA stimulates FANCL-catalyzed FANCD2 and FANCI monoubiquitylation. Collectively, these experiments identify RAD18-mediated PCNA monoubiquitination as a central hub for the mobilization of the FA pathway by promoting FANCL-mediated FANCD2 monoubiquitylation.     

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.     

Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.     

The WD40 repeats of FANCL are required for Fanconi anemia core complex assembly

Fanconi anemia (FA) is an autosomal recessive disorder characterized by aplastic anemia, cancer susceptibility, and cellular sensitivity to mitomycin C. Eight of the 11 cloned Fanconi anemia gene products (FANCA, -B, -C, -E, -F, -G, -L, and -M) form a multisubunit nuclear complex (FA core complex) required for monoubiquitination of a downstream FA protein, FANCD2. FANCL, which possesses three WD40 repeats and a plant homeodomain (PHD), is the putative E3 ubiquitin ligase subunit of the FA complex. Here, we demonstrate that the WD40 repeats of FANCL are required for interaction with other subunits of the FA complex. The PHD is dispensable for this interaction, although it is required for FANCD2 mono-ubiquitination. The PHD of FANCL also shares sequence similarity to the canonical RING finger of c-CBL, including a conserved tryptophan required for E2 binding by c-CBL. Mutation of this tryptophan in the FANCL PHD significantly impairs in vivo mono-ubiquitination of FANCD2 and in vitro auto-ubiquitination activity, and partially impairs restoration of mitomycin C resistance. We propose a model in which FANCL, via its WD40 region, binds the FA complex and, via its PHD, recruits an as-yet-unidentified E2 for mono-ubiquitination of FANCD2.     

FANCL Replaces BRCA1 as the Likely Ubiquitin Ligase Responsible for FANCD2 Monoubiquitination

Monoubiquitination of FANCD2 is a key step in the DNA damage response pathway involving Fanconi anemia proteins and the breast cancer susceptibility gene products, BRCA1 and BRCA2. One critical unresolved issue is the identity of the ubiquitin ligase responsible for this reaction. Two proteins, BRCA1 and FANCL(PHF9), have been suggested to be this ligase. Here we found that FANCL, but not BRCA1, evolutionarily co-exists with FANCD2 in several species. Moreover, the proportion of FANCD2 in chromatin and nuclear matrix is drastically reduced in a cell line mutated in FANCL, but not in that mutated in BRCA1. This defective distribution of FANCD2 in the FANCL-mutant cell line is likely due to its defective monoubiquitination, because the monoubiquitinated FANCD2 preferentially associates with chromatin and nuclear matrix, whereas non-ubiquitinated FANCD2 largely resides in the soluble fraction. Our data support the notion that FANCL, but not BRCA1, is the likely ligase for FANCD2 monoubiquitination.     

FANCL replaces BRCA1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination

Monoubiquitination of FANCD2 is a key step in the DNA damage response pathway involving Fanconi anemia proteins and the breast cancer susceptibility gene products, BRCA1 and BRCA2. One critical unresolved issue is the identity of the ubiquitin ligase responsible for this reaction. Two proteins, BRCA1 and FANCL(PHF9), have been suggested to be this ligase. Here we found that FANCL, but not BRCA1, evolutionarily co-exists with FANCD2 in several species. Moreover, the proportion of FANCD2 in chromatin and nuclear matrix is drastically reduced in a cell line mutated in FANCL, but not in that mutated in BRCA1. This defective distribution of FANCD2 in the FANCL-mutant cell line is likely due to its defective monoubiquitination, because the monoubiquitinated FANCD2 preferentially associates with chromatin and nuclear matrix, whereas non-ubiquitinated FANCD2 largely resides in the soluble fraction. Our data support the notion that FANCL, but not BRCA1, is the likely ligase for FANCD2 monoubiquitination.     

DNA robustly stimulates FANCD2 monoubiquitylation in the complex with FANCI

FANCI and FANCD2 form a complex, and play essential roles in the repair of interstrand DNA crosslinks (ICLs) by the Fanconi anemia (FA) pathway. FANCD2 is monoubiquitylated by the FA core complex, composed of 10 FA proteins including FANCL as the catalytic E3 subunit. FANCD2 monoubiquitylation can be reconstituted with purified minimal components, such as FANCI, E1, UBE2T (E2) and FANCL (E3) in vitro; however, its efficiency is quite low as compared to the in vivo monoubiquitylation of FANCD2. In this study, we found that various forms of DNA, such as single-stranded, double-stranded and branched DNA, robustly stimulated the FANCD2 monoubiquitylation in vitro up to a level comparable to its in vivo monoubiquitylation. This stimulation of the FANCD2 monoubiquitylation strictly required FANCI, suggesting that FANCD2 monoubiquitylation may occur in the FANCI-FANCD2 complex. A FANCI mutant that was defective in DNA binding was also significantly defective in FANCD2 monoubiquitylation in vitro. In the presence of 5' flapped DNA, a DNA substrate mimicking the arrested replication fork, about 70% of the input FANCD2 was monoubiquitylated, while less than 1% FANCD2 monoubiquitylation was observed in the absence of the DNA. Therefore, DNA may be the unidentified factor required for proper FANCD2 monoubiquitylation.     

FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway

The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100-deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA-BRCA DNA damage response network.     

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.     

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.     

FANCI is a second monoubiquitinated member of the Fanconi anemia pathway

Activation of the Fanconi anemia (FA) DNA damage-response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence-based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.     

Structural determinants of human FANCF protein that function in the assembly of a DNA damage signaling complex

Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-Fbox(Skp2) ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex.     

The Fanconi family adds a fraternal twin

The Fanconi anemia (FA) pathway plays an important role in DNA repair. In a recent issue of Cell, Smogorzewska et al. (2007) now demonstrate that FANCD2 has a paralog, FANCI. FANCI and FANCD2 form the "ID" complex that loads onto chromatin after DNA damage. Like FANCD2, monoubiquitination of FANCI requires the FA core complex. Importantly, FANCI and FANCD2 monoubiquitination is co-dependent, suggesting a novel mechanism in ubiquitin conjugation.     

FANCL在原始生殖细胞的形成和范可尼贫血中的功能研究

Fanconi氏贫血是一种罕见的常染色体隐性遗传性疾病,表现为进行性骨髓衰竭、先天性骨骼畸形和易患癌症等。Fanconi aremia（FA）病人细胞染色体自发不稳定,并对DNA交联剂如丝裂霉素C高度敏感。目前已发现11种FA蛋白参与形成了一种DNA损伤应答途径。新蛋白FANCL是FA复合物蛋白,作为E3连接酶催化FANCD2单一泛素化,泛素化FANCD2导向染色质与BRCA2相互作用,修复DNA损伤。FANCL、FANCC和FANCA等FA蛋白缺失造成生殖细胞缺失性不育,胚胎期生殖细胞中FA途径可能调控原始生殖细胞的增殖。FANCL和睾丸特异性蛋白质GGNBP1、GGNBP2以及OAZ3都与睾丸特异性蛋白质GGN1相互作用,形成睾丸特异性复合物,有可能在成年睾丸中影响精子生成。     

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.     

The Carboxyl Terminus of FANCE Recruits FANCD2 to the Fanconi Anemia (FA) E3 Ligase Complex to Promote the FA DNA Repair Pathway

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.