Deficiency in incisions produced by XPF at the site of a DNA interstrand cross-link in Fanconi anemia cells

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.     

FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure: Activation is not required for repair of Cr(VI)-induced DSBs

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by γ-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult.     

FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure: activation is not required for repair of Cr(VI)-induced DSBs

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by gamma-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult.     

Ubiquitin‐like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway

Ubiquitin and ubiquitin‐like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre‐mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5‐deficient cells display spliceosome‐independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5–FANCI binding, we generated separation‐of‐function mutants to demonstrate that key aspects of FA pathway function, including FANCI–FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5–FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.     

Ferulic acid prevents methylglyoxal-induced protein glycation,DNA damage,and apoptosis in pancreatic β-cells

Methylglyoxal (MG) can react with amino acids of proteins to induce protein glycation and consequently the formation of advanced glycation end-products (AGEs). Previous studies reported that ferulic acid (FA) prevented glucose-, fructose-, and ribose-induced protein glycation. In this study, FA (0.1–1 mM) inhibited MG-induced protein glycation and oxidative protein damage in bovine serum albumin (BSA). Furthermore, FA (0.0125–0.2 mM) protected against lysine/MG-mediated oxidative DNA damage, thereby inhibiting superoxide anion and hydroxyl radical generation during lysine and MG reaction. In addition, FA did not have the ability to trap MG. Finally, FA (0.1 mM) pretreatment attenuated MG-induced decrease in cell viability and prevented MG-induced cell apoptosis in pancreatic β-cells. The results suggest that FA is capable of protecting β-cells from MG-induced cell damage during diabetes.     

Fanconi Anemia Proteins and the S Phase Checkpoint

DNA interstrand crosslinks (ICLs) repair represents a formidable task for mammalian cells. Indeed, such DNA lesions, bridging both opposite DNA helices, function as a roadblock for every DNA transaction, in particular DNA replication. The eight Fanconi anemia (FA) proteins interact in a common pathway that is thought to be central in ICLs sensing/repair. Interestingly, FA cells, either mutated in one of the proteins composing the FA core complex or in the downstream FA protein FANCD2, exhibited a partial intra-S checkpoint defect in response to crosslinked DNA. Most importantly, the FA proteins work in the ATR-NBS1 branch of the ICL-induced checkpoint pathway as demonstrated by knocking-down CHK1 or MRE11 expression in a FA background. Even though our data disclose a clear functional role for the FA proteins in the intra-S checkpoint response it does not give a definite answer on what FA proteins do in this process and how they participate in the suppression/restart of DNA synthesis.It seems conceivable that FA proteins participate in the process involved in the recovery of stalled replication forks, a common event in proliferating cells, possibly ensuring correct replication fork repair by homologous recombination.     

DNA structure-induced recruitment and activation of the Fanconi anemia pathway protein FANCD2

The Fanconi anemia (FA) pathway proteins are thought to be involved in the repair of irregular DNA structures including those encountered by the moving replication fork. However, the nature of the DNA structures that recruit and activate the FA proteins is not known. Because FA proteins function within an extended network of proteins, some of which are still unknown, we recently established cell-free assays in Xenopus laevis egg extracts to deconstruct the FA pathway in a fully replication-competent context. Here we show that the central FA pathway protein, xFANCD2, is monoubiquitinated (xFANCD2-L) rapidly in the presence of linear and branched double-stranded DNA (dsDNA) structures but not single-stranded or Y-shaped DNA. xFANCD2-L associates with dsDNA structures in an FA core complex-dependent manner but independently of xATRIP, the regulatory subunit of xATR. Formation of xFANCD2-L is also triggered in response to circular dsDNA, suggesting that dsDNA ends are not required to trigger monoubiquitination of FANCD2. The induction of xFANCD2-L in response to circular dsDNA is replication and checkpoint independent. Our results provide new evidence that the FA pathway discriminates among DNA structures and demonstrate that triggering the FA pathway can be uncoupled from DNA replication and ATRIP-dependent activation.     

Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair

Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.     

Fanconi anemia: at the Crossroads of DNA repair

Fanconi anemia (FA) is an autosomal disorder that causes genome instability. FA patients suffer developmental abnormalities, early-onset bone marrow failure, and a predisposition to cancer. The disease is manifested by defects in DNA repair, hypersensitivity to DNA crosslinking agents, and a high degree of chromosomal aberrations. The FA pathway comprises 13 disease-causing genes involved in maintaining genomic stability. The fast pace of study of the novel DNA damage network has led to the constant discovery of new FA-like genes involved in the pathway that when mutated lead to similar disorders. A majority of the FA proteins act as signal transducers and scaffolding proteins to employ other pathways to repair DNA. This review discusses what is known about the FA proteins and other recently linked FA-like proteins. The goal is to clarify how the proteins work together to carry out interstrand crosslink repair and homologous recombination-mediated repair of damaged DNA.     

Fanconi anemia proteins stabilize replication forks

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.     

3R coordination by Fanconi anemia proteins

Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow failure and hypersensitivity to DNA crosslinks. Nine FA genes have been isolated so far. The biochemical function(s) of the FA proteins remain(s) poorly determined. However, a large consensus exists on the evidence that, to cope with DNA cross-links, a cell needs a functional FA pathway. In this review, we resume current understanding of how the FA pathway works in response to DNA damage and how it is integrated in a complex network of proteins involved in the maintenance of the genetic stability.     

Loss of the Fanconi anemia group C protein activity results in an inability to activate caspase-3 after ionizing radiation

Fanconi anemia (FA) is a human genetic disease featuring cancer predisposition, genetic instability and DNA damage hypersensitivity. Although abnormalities in DNA repair and cell cycle checkpoint have been proposed as the underlying defect in this syndrome, these hypotheses did not provide full explanations of the complex phenotype. Although not exclusive of such possibilities, alterations in the control of apoptosis might account for the pleiotropic phenotype of this syndrome. We and others have previously reported a deregulation of the apoptotic response to mitomycin C, suggesting that the products of the Fanconi anemia group C protein (FANCC) contribute to the regulation of apoptosis. To explore the functional importance of the apoptotic alterations in FA we analyzed biochemical steps of the execution phase of apoptosis stimulated by another DNA damaging agent, the gamma-ray using FA cell lines derived from complementation group C (FA-C) independent patients. It is shown that the poly(ADP-ribose) polymerase, a target of caspase-3, is not cleaved in FA-C after ionizing radiation (IR). Moreover, caspase-3 is not processed in its active form and, its activity is not increased by IR in FA-C cells compared to normal cells. Altogether, these results demonstrate that loss of the FANCC activity results in a deficiency of the IR-induced apoptosis which is due to an inability to activate caspase-3. Our work suggests that apoptosis signaling induced by mitomycin C and IR is subject to common regulation involving the FANCC protein.     

What is wrong with Fanconi anemia cells?

Figuring out what is wrong in Fanconi anemia (FA) patient cells is critical to understanding the contributions of the FA pathway to DNA repair and tumor suppression. Although FA patients exhibit a wide range of disease manifestation as well as severity (asymptomatic to congenital abnormalities, bone marrow failure, and cancer), cells from FA patients share underlying defects in their ability to process DNA lesions that interfere with DNA replication. In particular, FA cells are very sensitive to agents that induce DNA interstrand crosslinks (ICLs). The cause of this pronounced ICL sensitivity is not fully understood, but has been linked to the aberrant activation of DNA damage repair proteins, checkpoints and pathways. Thus, regulation of these responses through coordination of repair processing at stalled replication forks is an essential function of the FA pathway. Here, we briefly summarize some of the aberrant DNA damage responses contributing to defects in FA cells, and detail the newly-identified relationship between FA and the mismatch repair protein, MSH2. Understanding the contribution of MSH2 and/or other proteins to the replication problem in FA cells will be key to assessing therapeutic options to improve the health of FA patients. Moreover, loss of these factors, if linked to improved replication, could be a key event in the progression of FA cells to cancer cells. Likewise, loss of these factors could synergize to enhance tumorigenesis or confer chemoresistance in tumors defective in FA-BRCA pathway proteins and provide a basis for biomarkers for disease progression and response.     

The Fanconi anemia/BRCA pathway: a coordinator of cross-link repair

Fanconi anemia (FA) is a rare inherited disease characterized by genomic instability and markedly increased cancer risk. Efforts to elucidate the molecular basis of FA have unearthed a novel DNA damage response pathway, the integrity of which is critical for cellular resistance to DNA cross-linking agents. Despite significant progress in uncovering the molecular events underlying FA, the precise function of this pathway in DNA repair is unknown. This article will review evidence implicating FA proteins in multiple aspects of DNA cross-link repair and propose a model to explain the selectivity of the FA pathway toward DNA cross-linking agents.     

The Fanconi anemia pathway and the DNA interstrand cross-links repair

Fanconi anemia (FA) is a genetic cancer-predisposition syndrome characterized by bone marrow failure and cellular and chromosomal hypersensitivity to DNA cross-linking agents. Seven FA genes have been isolated and their products associate to form a pathway that interacts functionally or physically with several DNA-damage response proteins involved in cell cycle checkpoints and/or DNA repair. These proteins include BLM, ATM, BRCA1, XPF and the MRE11/RAD50/NBS1 complex. In spite of several recent striking progresses in the biochemistry and the molecular biology of the disorder, the precise function(s) of the FA proteins remain(s) poorly determined. However, several recent data indicate that the FA pathway could be involved in the coordination of both cell cycle checkpoints and DNA repair.     

FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability

FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.     

Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedreich's ataxia

Respiratory chain dysfunction has been identified in several neurodegenerative disorders. In Friedreich's ataxia (FA) and Huntington's disease (HD), where the respective mutations are in nuclear genes encoding non-respiratory chain mitochondrial proteins, the defects in oxidative phosphorylation are clearly secondary. In Parkinson's disease (PD) the situation is less clear, with some evidence for a primary role of mitochondrial DNA in at least a proportion of patients. The pattern of the respiratory chain defect may provide some clue to its cause; in PD there appears to be a selective complex I deficiency; in HD and FA the deficiencies are most severe in complex II/III with a less severe defect in complex IV. Aconitase activity in HD and FA is severely decreased in brain and muscle, respectively, but appears to be normal in PD brain. Free radical generation is thought to be of importance in both HD and FA, via excitotoxicity in HD and abnormal iron handling in FA. The oxidative damage observed in PD may be secondary to the mitochondrial defect. Whatever the cause(s) and sequence of events, respiratory chain deficiencies appear to play an important role in the pathogenesis of neurodegeneration. The mitochondrial abnormalities induced may converge on the function of the mitochondrion in apoptosis. This mode of cell death is thought to play an important role in neurodegenerative diseases and it is tempting to speculate that the observed mitochondrial defects in PD, HD and FA result directly in apoptotic cell death, or in the lowering of a cell's threshold to undergo apoptosis. Clarifying the role of mitochondria in pathogenesis may provide opportunities for the development of treatments designed to reverse or prevent neurodegeneration.     

Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks

Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.     

The DNA crosslink-induced S-phase checkpoint depends on ATR-CHK1 and ATR-NBS1-FANCD2 pathways

The genetic syndrome Fanconi anemia (FA) is characterized by aplastic anemia, cancer predisposition and hypersensitivity to DNA interstrand crosslinks (ICLs). FA proteins (FANCs) are thought to work in pathway(s) essential for dealing with crosslinked DNA. FANCs interact with other proteins involved in both DNA repair and S-phase checkpoint such as BRCA1, ATM and the RAD50/MRE11/NBS1 (RMN) complex. We deciphered the previously undefined pathway(s) leading to the ICLs-induced S-phase checkpoint and the role of FANCs in this process. We found that ICLs activate a branched pathway downstream of the ATR kinase: one branch depending on CHK1 activity and the other on the FANCs-RMN complex. The transient slow-down of DNA synthesis was abolished in cells lacking ATR, whereas CHK1-siRNA-treated cells, NBS1 or FA cells showed partial S-phase arrest. CHK1 RNAi in NBS1 or FA cells abolished the S-phase checkpoint, suggesting that CHK1 and FANCs/NBS1 proteins work on parallel pathways. Furthermore, we found that ICLs trigger ATR-dependent FANCD2 phosphorylation and FANCD2/ATR colocalization. This study demonstrates a novel relationship between the FA pathway(s) and the ATR kinase.     

A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome

Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIalpha and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.