FANCD2-Controlled Chromatin Access of the Fanconi-Associated Nuclease FAN1 Is Crucial for the Recovery of Stalled Replication Forks

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2^Ub) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2^Ub isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation.     

The conserved Fanconi anemia nuclease Fan1 and the SUMO E3 ligase Pli1 act in two novel Pso2-independent pathways of DNA interstrand crosslink repair in yeast

DNA interstrand cross-links (ICLs) represent a physical barrier to the progression of cellular machinery involved in DNA metabolism. Thus, this type of adduct represents a serious threat to genomic stability and as such, several DNA repair pathways have evolved in both higher and lower eukaryotes to identify this type of damage and restore the integrity of the genetic material. Human cells possess a specialized ICL-repair system, the Fanconi anemia (FA) pathway. Conversely yeasts rely on the concerted action of several DNA repair systems. Recent work in higher eukaryotes identified and characterized a novel conserved FA component, FAN1 (Fanconi anemia-associated nuclease 1, or FANCD2/FANCI-associated nuclease 1). In this study, we characterize Fan1 in the yeast Schizosaccharomyces pombe. Using standard genetics, we demonstrate that Fan1 is a key component of a previously unidentified ICL-resolution pathway. Using high-throughput synthetic genetic arrays, we also demonstrate the existence of a third pathway of ICL repair, dependent on the SUMO E3 ligase Pli1. Finally, using sequence-threaded homology models, we predict and validate key residues essential for Fan1 activity in ICL repair.     

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.     

Crosstalk between BRCA‐Fanconi anemia and mismatch repair pathways prevents MSH2‐dependent aberrant DNA damage responses

Several proteins in the BRCA‐Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA‐FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA‐FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18‐dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2‐null primary mouse cells. Thus, we propose that regulation of MSH2‐dependent DNA damage response underlies the importance of interactions between BRCA‐FA and MMR pathways.     

The nuclease FAN1 is involved in DNA crosslink repair in Arabidopsis thaliana independently of the nuclease MUS81

Fanconi anemia is a severe genetic disorder. Mutations in one of several genes lead to defects in DNA crosslink (CL) repair in human cells. An essential step in CL repair is the activation of the pathway by the monoubiquitination of the heterodimer FANCD2/FANCI, which recruits the nuclease FAN1 to the CL site. Surprisingly, FAN1 function is not conserved between different eukaryotes. No FAN1 homolog is present in Drosophila and Saccharomyces cerevisiae. The FAN1 homolog in Schizosaccharomyces pombe is involved in CL repair; a homolog is present in Xenopus but is not involved in CL repair. Here we show that a FAN1 homolog is present in plants and it is involved in CL repair in Arabidopsis thaliana. Both the virus-type replication-repair nuclease and the ubiquitin-binding ubiquitin-binding zinc finger domains are essential for this function. FAN1 likely acts upstream of two sub-pathways of CL repair. These pathways are defined by the Bloom syndrome homolog RECQ4A and the ATPase RAD5A, which is involved in error-free post-replicative repair. Mutations in both FAN1 and the endonuclease MUS81 resulted in greater sensitivity against CLs than in the respective single mutants. These results indicate that the two nucleases define two independent pathways of CL repair in plants.     

Structural and functional relationships of FAN1

FANCD2/FANCI-associated nuclease (FAN1) is a 5′ flap structure-specific endonuclease and 5′ to 3′ exonuclease. This nuclease can resolve interstrand cross-links (ICLs) independently of the Fanconi anemia (FA) pathway and controls the progression of stalled replication forks in an FA-dependent manner, thereby maintaining chromosomal stability. Several FAN1 mutations are observed in various cancers and degenerative diseases. Recently, several crystal structures of the FAN1-DNA complexes have been reported, and to date, these represent the only structures for a DNA bound ICL-repair nuclease. Puzzlingly, human FAN1 forms two different quaternary structures with different DNA binding modes, and based on these structures, two ICL-repair mechanisms have been proposed. In one mechanism, monomeric FAN1 recognizes the 5′ flap terminal phosphate via a basic pocket and successively cleaves at every third nucleotide of the DNA substrates. In the other mechanism, dimeric FAN1 scans, latches, and unwinds the postnick duplex of the substrate DNA to direct the scissile phosphodiester group to the active site. In this review, we discuss the structures, function, and proposed mechanisms of FAN1 nuclease, and provide the insights into its role in ICL repair and in processing of stalled replication forks.     

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。     

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.     

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.     

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions.     

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.     

DNA2 and EXO1 in replication-coupled,homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.     

DNA2 and EXO1 in replication-coupled,homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.     

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.     

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.     

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.     

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.     

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.     

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。