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.     

Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM

The Fanconi anemia (FA) core complex plays a crucial role in a DNA damage response network with BRCA1 and BRCA2. How this complex interacts with damaged DNA is unknown, as only the FA core protein FANCM (the homolog of an archaeal helicase/nuclease known as HEF) exhibits DNA binding activity. Here, we describe the identification of FAAP24, a protein that targets FANCM to structures that mimic intermediates formed during the replication/repair of damaged DNA. FAAP24 shares homology with the XPF family of flap/fork endonucleases, associates with the C-terminal region of FANCM, and is a component of the FA core complex. FAAP24 is required for normal levels of FANCD2 monoubiquitylation following DNA damage. Depletion of FAAP24 by siRNA results in cellular hypersensitivity to DNA crosslinking agents and chromosomal instability. Our data indicate that the FANCM/FAAP24 complex may play a key role in recruitment of the FA core complex to damaged DNA.     

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.     

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.     

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.     

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。     

Human MutS and FANCM complexes function as redundant DNA damage sensors in the Fanconi Anemia pathway

The Fanconi Anemia (FA) pathway encodes a DNA damage response activated by DNA damage-stalled replication forks. Current evidence suggests that the FA pathway initiates with DNA damage recognition by the FANCM complex (FANCM/FAAP24/MHF). However, genetic inactivation of FANCM in mouse and DT40 cells causes only a partial defect in the FA pathway activation, suggesting the existence of redundant DNA damage sensors. Here we show that the MutS homologs function in this capacity. A RNAi screen revealed that MSH2 silencing caused defective FA pathway activation, as assessed by damage-induced FANCD2 mono-ubiquitination. A similar FA pathway defect was observed with MSH3 or MSH6 silencing. MSH2 depletion caused cellular phenotypes associated with defective FA pathway, including mitomycin C hypersensitivity and chromosomal instability. Further, silencing of FANCM in MSH2 deficient HEC59 cells caused a more severe FA defect relative to comparable silencing in MSH2 complemented HEC59 + Chr2 cells, suggesting redundant functions between MSH2 and FANCM. Consistent with this hypothesis, depletion of MSH2 resulted in defective chromatin localization of the FA core complex upon DNA damage. Further, MSH2 was co-purified and co-immunoprecipitated with FA core complex components. Taken together, our results suggest that human MutS homologs and FANCM complexes function as redundant DNA damage sensors of the FA pathway.     

FANCM and FAAP24 function in ATR-mediated checkpoint signaling independently of the Fanconi anemia core complex

The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.     

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Genomic stability requires a functional Fanconi anemia (FA) pathway composed of an upstream “core complex” (FA proteins A/B/C/E/F/G/L/M) that mediates monoubiquitination of the downstream targets FANCD2 and FANCI. Unique among FA core complex members, FANCM has processing activities toward replication-associated DNA structures, suggesting a vital role for FANCM during replication. Using Xenopus egg extracts, we analyzed the functions of FANCM in replication and the DNA damage response. xFANCM binds chromatin in a replication-dependent manner and is phosphorylated in response to DNA damage structures. Chromatin binding and DNA damage-induced phosphorylation of xFANCM are mediated in part by the downstream FA pathway protein FANCD2. Moreover, phosphorylation and chromatin recruitment of FANCM is regulated by two mayor players in the DNA damage response: the cell cycle checkpoint kinases ATR and ATM. Our results indicate that functions of FANCM are controlled by FA- and non-FA pathways in the DNA damage response.Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Cells from FA³ patients show hypersensitivity to DNA interstrand cross-links and have highly elevated chromosomal breakage rates, indicating a role for FA proteins in the cellular DNA damage response. The FA pathway consists of an upstream FA core complex containing at least eight proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) that is required for the DNA damage-induced monoubiquitination of two downstream proteins, FANCD2 and FANCI. Although the molecular function of the FA pathway is unknown, the identification of additional FA genes FANCD1 (BRCA2), FANCN (PALB2), and the DNA helicase FANCJ (BRIP1) as breast cancer (BRCA) susceptibility genes suggests convergence of the FA/BRCA pathway with a larger network of proteins involved in DNA repair (reviewed in Ref. 1).In addition to monoubiquitination by the FA core complex, FANCD2 and FANCI are phosphorylated by the two major cell cycle checkpoint kinases, ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related),y in response to DNA damage (2–6). ATM-dependent phosphorylation of FANCD2 occurs following ionizing irradiation and is required for activation of the ionizing irradiation-induced intra-S phase checkpoint (4). ATR-dependent phosphorylation of FANCD2 is triggered by various types of DNA damage, including replication stress, and is required for the interstrand cross-link-induced intra-S phase checkpoint response (2, 3). Moreover, phosphorylation by ATR is required for efficient FANCD2 monoubiquitination in response to DNA damage, suggesting that the FA pathway might participate in ATR-dependent coordination of the S phase of the cell cycle (3, 7).The recent identification of a highly conserved FA core complex member, FANCM (8, 9), indicates a direct role of FA pathway proteins in repair steps at sites of DNA damage. FANCM is a homolog of the archaebacterial Hef protein (helicase-associated endonuclease for fork-structured DNA) and contains two DNA processing domains: a DEAH box helicase domain and an XPF/ERCC4-like endonuclease domain. FANCM has ATP-dependent DNA translocase activity and can dissociate DNA triple helices in vitro (8). Moreover, FANCM binds Holliday junctions and DNA replication fork structures in vitro and promotes ATP-dependent branch point migration, suggesting that FANCM might be involved in DNA processing at stalled replication forks (10, 11). In human cells, FANCM localizes to chromatin and is required for chromatin recruitment of other FA core complex proteins (8, 12). FANCM is phosphorylated during both the M and S phases and in response to DNA-damaging agents (8, 12, 13). Interestingly, DNA damage-induced phosphorylation of FANCM is independent of the FA core complex (8), suggesting that FANCM is controlled by other, as yet unknown upstream components of the DNA damage response. Here, we used cell-free Xenopus egg extracts to investigate the role of FANCM during replication and in the DNA damage response. We show that Xenopus FANCM (xFANCM) binds chromatin in a replication-dependent manner and is phosphorylated during unperturbed replication as well as in response to various DNA damage structures. Both chromatin recruitment and phosphorylation of xFANCM are partially controlled by xFANCD2, suggesting feedback signaling from xFANCD2 to the upstream xFA core complex via regulation of xFANCM. In addition, chromatin recruitment during unperturbed replication and activation of xFANCM in response to DNA damage are controlled by the xATR and xATM cell cycle kinases.     

BRCA1-independent ubiquitination of FANCD2

Monoubiquitination of the FANCD2 protein is a key step in the Fanconi anemia (FA) tumor suppressor pathway, coinciding with this molecule's accumulation at sites of genome damage. Strong circumstantial evidence points to a requirement for the BRCA1 gene product in this step. Here, we show that the purified BRCA1/BARD1 complex, together with E1 and UbcH5a, is sufficient to reconstitute the monoubiquitination of FANCD2 in vitro. Although siRNA-mediated knockdown of BRCA1 in human cells results in defective targeting of FANCD2 to sites of DNA damage, it does not lead to a defect in FANCD2 ubiquitination. Furthermore, ablation of the RING finger domains of either BRCA1 or BARD1 in the chicken B cell line DT40 also leaves FANCD2 modification intact. Consequently, while BRCA1 affects the accumulation of FANCD2 at sites of DNA damage, BRCA1/BARD1 E3 ligase activity is not essential for the monoubiquitination of FANCD2.     

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.     

Chk1-mediated phosphorylation of FANCE is required for the Fanconi anemia/BRCA pathway

The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.     

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.     

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.     

The FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during DNA double-strand break repair

The Fanconi anemia (FA) core complex promotes the tolerance/repair of DNA damage at stalled replication forks by catalyzing the monoubiquitination of FANCD2 and FANCI. Intriguingly, the core complex component FANCM also catalyzes branch migration of model Holliday junctions and replication forks in vitro. Here we have characterized the ortholog of FANCM in fission yeast Fml1 in order to understand the physiological significance of this activity. We show that Fml1 has at least two roles in homologous recombination-it promotes Rad51-dependent gene conversion at stalled/blocked replication forks and limits crossing over during mitotic double-strand break repair. In vitro Fml1 catalyzes both replication fork reversal and D loop disruption, indicating possible mechanisms by which it can fulfill its pro- and antirecombinogenic roles.     

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。     

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.     

UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation

The Fanconi anemia pathway is required for the efficient repair of damaged DNA. A key step in this pathway is the monoubiquitination of the FANCD2 protein by the ubiquitin ligase (E3) composed of Fanconi anemia core complex proteins. Here, we show that UBE2T is the ubiquitin-conjugating enzyme (E2) essential for this pathway. UBE2T binds to FANCL, the ubiquitin ligase subunit of the Fanconi anemia core complex, and is required for the monoubiquitination of FANCD2 in vivo. DNA damage in UBE2T-depleted cells leads to the formation of abnormal chromosomes that are a hallmark of Fanconi anemia. In addition, we show that UBE2T undergoes automonoubiquitination in vivo. This monoubiquitination is stimulated by the presence of the FANCL protein and inactivates UBE2T. Therefore, UBE2T is the E2 in the Fanconi anemia pathway and has a self-inactivation mechanism that could be important for negative regulation of the Fanconi anemia 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.