Hsp90 and the Fanconi Anemia Pathway: A Molecular Link Between Protein Quality Control and the DNA Damage Response

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an essential role in cell growth and survival. The chaperone exerts these functions by regulating key signaling proteins involved in cell growth/survival and protecting cells from proteotoxic stress. Importantly, Hsp90 inhibitors including geldanamycin analogues show anti-tumor effects. We recently found that Hsp90 promotes stabilization and nuclear localization of the Fanconi anemia (FA) protein FANCA, which is required for activation of the FA pathway. The FA pathway is a multiprotein biochemical pathway involved in genotoxic signaling, defects in which cause genomic instability, hematopoietic stem cell failure and tumor development. Inhibition of Hsp90 impairs the intracellular homeostasis of FANCA, resulting in disruption of the FA pathway. These findings have important implications for rational cancer chemotherapy using Hsp90 inhibitors. We also discuss the possible functions of Hsp90 in FA pathophysiology and stem cell/cancer biology. Based on our findings and other data, we propose that Hsp90 functions as “a guardian of the genome” through the control of DNA repair proteins.     

Cooperation of the ATM and Fanconi Anemia/BRCA Pathways in Double-Strand Break End Resection

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FANCD2, FANCJ and BRCA2 cooperate to promote replication fork recovery independently of the Fanconi Anemia core complex

Fanconi Anemia (FA) is an inherited multi-gene cancer predisposition syndrome that is characterized on the cellular level by a hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA pathway proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex monoubiquitinates the central FA pathway members FANCD2 and FANCI, followed by their recruitment to chromatin. Chromatin-bound monoubiquitinated FANCD2 and FANCI subsequently coordinate DNA repair factors including the downstream FA pathway members FANCJ and FANCD1/BRCA2 to repair the DNA ICL. Importantly, we recently showed that FANCD2 has additional independent roles: it binds chromatin and acts in concert with the BLM helicase complex to promote the restart of aphidicolin (APH)-stalled replication forks, while suppressing the firing of new replication origins. Here, we show that FANCD2 fulfills these roles independently of the FA core complex-mediated monoubiquitination step. Following APH treatment, nonubiquitinated FANCD2 accumulates on chromatin, recruits the BLM complex, and promotes robust replication fork recovery regardless of the absence or presence of a functional FA core complex. In contrast, the downstream FA pathway members FANCJ and BRCA2 share FANCD2's role in replication fork restart and the suppression of new origin firing. Our results support a non-linear FA pathway model at stalled replication forks, where the nonubiquitinated FANCD2 isoform – in concert with FANCJ and BRCA2 – fulfills a specific function in promoting efficient replication fork recovery independently of the FA core complex.     

FANCD2, FANCJ and BRCA2 cooperate to promote replication fork recovery independently of the Fanconi Anemia core complex

Fanconi Anemia (FA) is an inherited multi-gene cancer predisposition syndrome that is characterized on the cellular level by a hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA pathway proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex monoubiquitinates the central FA pathway members FANCD2 and FANCI, followed by their recruitment to chromatin. Chromatin-bound monoubiquitinated FANCD2 and FANCI subsequently coordinate DNA repair factors including the downstream FA pathway members FANCJ and FANCD1/BRCA2 to repair the DNA ICL. Importantly, we recently showed that FANCD2 has additional independent roles: it binds chromatin and acts in concert with the BLM helicase complex to promote the restart of aphidicolin (APH)-stalled replication forks, while suppressing the firing of new replication origins. Here, we show that FANCD2 fulfills these roles independently of the FA core complex-mediated monoubiquitination step. Following APH treatment, nonubiquitinated FANCD2 accumulates on chromatin, recruits the BLM complex, and promotes robust replication fork recovery regardless of the absence or presence of a functional FA core complex. In contrast, the downstream FA pathway members FANCJ and BRCA2 share FANCD2''s role in replication fork restart and the suppression of new origin firing. Our results support a non-linear FA pathway model at stalled replication forks, where the nonubiquitinated FANCD2 isoform – in concert with FANCJ and BRCA2 – fulfills a specific function in promoting efficient replication fork recovery independently of the FA core complex.     

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.     

Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiquitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation-induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.     

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.     

TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response

Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response.     

DNA Damage Response as an Anti-Cancer Barrier: Damage Threshold and the Concept of 'Conditional Haploinsufficiency'

DNA damage response (DDR) emerges as a biological tumorigenesis barrier in early stages of cancer development, and a selective pressure that favors outgrowth of malignant clones with defects in the genome maintenance machinery, such as mutations of p53 and other DDR components. Recent studies indicate that the DDR barrier is not alarmed universally among early noninvasive lesions, but rather responds to high-risk tumorigenic threats that occur in high-grade, pre-malignant lesions that are generally more likely to develop into bona fide malignancies. In addition, while the DDR barrier appears to operate in major types of cancer, such as carcinomas of the lung, breast and colon, DDR activation is rare at any stage of progression among testicular germ-cell tumors. Together with observations that several, but not all oncogenic insults are capable of activating the DDR machinery, these new results point to existence of a critical threshold of such oncogene-induced DNA damage. It seems that only cells and lesions that experience DNA replication stress and DNA damage above such threshold activate the cellular senescence or cell death pathways within the DDR machinery. The higher load of DNA damage may also contribute to cancer predisposition in families with inherited heterozygous defects in the DDR barrier, such as in ATM, BRCA1, BRCA2, p53 and other genes. We propose that carriers of such DDR defects may be more prone to malignancy due to ‘conditional haploinsufficiency’: such partial defects may be asymptomatic in normal tissues, yet they may become manifest under conditions of supra-threshold endogenous DNA damage in oncogene-driven pre-malignant lesions.     

DNA damage response and cancer therapeutics through the lens of the Fanconi Anemia DNA repair pathway

Fanconi Anemia (FA) is a rare, inherited genomic instability disorder, caused by mutations in genes involved in the repair of interstrand DNA crosslinks (ICLs). The FA signaling network contains a unique nuclear protein complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and coordinates activities of the downstream DNA repair pathway including nucleotide excision repair, translesion synthesis, and homologous recombination. FA proteins act at different steps of ICL repair in sensing, recognition and processing of DNA lesions. The multi-protein network is tightly regulated by complex mechanisms, such as ubiquitination, phosphorylation, and degradation signals that are critical for the maintenance of genome integrity and suppressing tumorigenesis. Here, we discuss recent advances in our understanding of how the FA proteins participate in ICL repair and regulation of the FA signaling network that assures the safeguard of the genome. We further discuss the potential application of designing small molecule inhibitors that inhibit the FA pathway and are synthetic lethal with DNA repair enzymes that can be used for cancer therapeutics.     

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

FANCM-FAAP24 and HCLK2: Roles in ATR signalling and the Fanconi Anemia pathway

The ATR signalling pathway coordinates the cellular response to replication stress, which is essential for the maintenance of genome integrity. HCLK2/Tel2 is a highly conserved orphan protein that binds directly to ATR and other PI3-kinase related kinases and plays a central role in checkpoint signalling responses. Proteomic analyses of HCLK2 complexes confirmed ATR, ATRIP and DNA-PKcs as HCLK2 interacting factors and also uncovered two surprising interacting proteins, the heterodimeric Fanconi Anemia (FA) proteins FANCM and FAAP24. Our subsequent findings that ATR signalling is attenuated in FANCM and FAAP24-depleted cells, together with recent biochemical studies, suggested that remodelling of stalled replication forks by FANCM-FAAP24 is required to facilitate efficient activation of ATR signalling in response to replication stress. Furthermore, our study revealed that the DNA translocase activity of FANCM is essential for efficient activation of the ATR signalling, a function that is separate and distinct from its role in targeting the FA core complex to sites of DNA damage. In this review we discuss the importance of these findings in the context of recent data and raise questions regarding the role of HCLK2 and FANCM-FAAP24 in human disease.