Structure of the Fanconi anaemia monoubiquitin ligase complex.

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.

Arsenite Binds to the RING Finger Domain of FANCL E3 Ubiquitin Ligase and Inhibits DNA Interstrand Crosslink Repair.

Human exposure to arsenic in drinking water is known to be associated with the development of bladder, lung, kidney, and skin cancers. The molecular mechanisms underlying the carcinogenic effects of arsenic species remain incompletely understood. DNA interstrand cross-links (ICLs) are among the most cytotoxic type of DNA lesions that block DNA replication and transcription, and these lesions can be induced by endogenous metabolism and by exposure to exogenous agents. Fanconi anemia (FA) is a congenital disorder manifested with elevated sensitivity toward DNA interstrand cross-linking agents, and monoubiquitination of FANCD2 by FANCL is a crucial step in FA-mediated DNA repair. Here, we demonstrated that As3+ could bind to the PHD/RING finger domain of FANCL in vitro and in cells. This binding led to compromised ubiquitination of FANCD2 in cells and diminished recruitment of FANCD2 to chromatin and DNA damage sites induced by 4,5',8-trimethylpsoralen plus UVA irradiation. Furthermore, clonogenic survival assay results showed that arsenite coexposure rendered cells more sensitive toward DNA interstrand cross-linking agents. Together, our study suggested that arsenite may compromise genomic stability via perturbation of the Fanconi anemia pathway, thereby conferring its carcinogenic effect.

BLM promotes the activation of Fanconi Anemia signaling pathway.

Mutations in the human RecQ helicase, BLM, causes Bloom Syndrome, which is a rare autosomal recessive disorder and characterized by genomic instability and an increased risk of cancer. Fanconi Anemia (FA), resulting from mutations in any of the 19 known FA genes and those yet to be known, is also characterized by chromosomal instability and a high incidence of cancer. BLM helicase and FA proteins, therefore, may work in a common tumor-suppressor signaling pathway. To date, it remains largely unclear as to how BLM and FA proteins work concurrently in the maintenance of genome stability. Here we report that BLM is involved in the early activation of FA group D2 protein (FANCD2). We found that FANCD2 activation is substantially delayed and attenuated in crosslinking agent-treated cells harboring deficient Blm compared to similarly treated control cells with sufficient BLM. We also identified that the domain VI of BLM plays an essential role in promoting FANCD2 activation in cells treated with DNA crosslinking agents, especially ultraviolet B. The similar biological effects performed by ΔVI-BLM and inactivated FANCD2 further confirm the relationship between BLM and FANCD2. Mutations within the domain VI of BLM detected in human cancer samples demonstrate the functional importance of this domain, suggesting human tumorigenicity resulting from mtBLM may be at least partly attributed to mitigated FANCD2 activation. Collectively, our data show a previously unknown regulatory liaison in advancing our understanding of how the cancer susceptibility gene products act in concert to maintain genome stability.

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.

The genetic and biochemical basis of FANCD2 monoubiquitination.

Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.

Regulation of FANCD2 and FANCI monoubiquitination by their interaction and by DNA.

FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.

Structure of the human FANCL RING-Ube2T complex reveals determinants of cognate E3-E2 selection.

The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ∼40 E2s and ∼600 E3s giving rise to a possible ∼24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL.

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.

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.

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.

The structure of the catalytic subunit FANCL of the Fanconi anemia core complex.

The Fanconi anemia (FA) pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the FA core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the FA core complex, at 3.2 A. The structure reveals an architecture fundamentally different from previous sequence-based predictions. The molecule is composed of an N-terminal E2-like fold, which we term the ELF domain, a novel double-RWD (DRWD) domain, and a C-terminal really interesting new gene (RING) domain predicted to facilitate E2 binding. Binding assays show that the DRWD domain, but not the ELF domain, is responsible for substrate binding.

Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI.

A key step in the Fanconi anemia (FA) tumor suppressor pathway is the site-specific monoubiquitination of the FANCD2 protein. Genetic studies indicate that this crucial modification requires eight known FA gene products and the E2-conjugating enzyme Ube2t. Here, we minimally reconstitute this monoubiquitination reaction with Ube2t and the FANCL protein, revealing that monoubiquitination is stimulated by a conserved RWD-like domain in FANCL. Furthermore, addition of the FANCI protein enhances monoubiquitination and also restricts it to the in vivo substrate lysine residue on FANCD2. This work therefore establishes a system that provides mechanistic insight into the functions of FANCL and FANCI in the catalysis of FANCD2 monoubiquitination.

A requirement of FancL and FancD2 monoubiquitination in DNA repair.

The rare hereditary disorder Fanconi anemia (FA) can be caused by mutations in components of the FA core complex (FancA/B/C/E/F/G/L/M), a key regulator FancD2, the breast cancer susceptibility protein BRCA2/FancD1, or the newly identified FancJ/BRIP1 helicase. By performing yeast two-hybrid (Y2H) screens using N-terminal chicken (ch) FancD2 as a bait, we have identified chFancL, the likely ubiquitin E3 ligase subunit of the FA core complex. We also found that ectopically expressed FancD2 and FancL co-immunoprecipitated in 293T cells, and this interaction was dependent on the PHD domain of FancL. FANCL-disrupted chicken DT40 cells displayed defects in both FancD2 monoubiquitination and focus formation. Importantly, cell lines lacking the FANCL or FANCD2 genes, or carrying a "knock-in" mutation of the FancD2 monoubiquitination site (where the Lys 563 residue is changed to Arg), displayed quantitatively identical defects in the repair of I-SceI-induced chromosomal breaks by homologous recombination (HR). These data establish the role of FANCL and FancD2 monoubiquitination in HR repair.

Evidence for subcomplexes in the Fanconi anemia pathway.

Fanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur "upstream" in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2.

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.