FANCD2 Foci Research Articles

Fanconi Anemia pathway heterogeneity revealed by cisplatin and oxaliplatin treatments

Genetic or epigenetic inactivation of the pathway formed by the Fanconi Anemia (FA) proteins occurs in several cancer types, including head and neck squamous cell carcinomas (HNSCC), rendering the affected tumors potentially hypersensitive to DNA crosslinking agents. However, the cytotoxicity of other commonly used cancer therapeutics in cells with FA pathway defects remains to be defined. Here, we focused on the effects of cisplatin and oxaliplatin in a panel of HNSCC and fibroblast cell lines. We found that FANCC- and FANCD2-mutant cells were unexpectedly more sensitive to platinum drugs than FANCA-mutant cells, and mono-ubiquitination of FANCD2, which is mediated by the FANCA and FANCC containing FA core complex was not required for platinum resistance. Interestingly, platinum hypersensitivity could be dissociated from mitomycin C hypersensitivity suggesting different underlying mechanisms. FANCD2 or RAD51 subnuclear foci were not useful as biomarkers of platinum hypersensitivity of FANCC/FANCD2-mutant cells. Our data add to an emerging body of evidence indicating that the FA pathway is not linear and that several protein subcomplexes with different functions exist. It will be important to establish biomarkers that can predict the sensitivity of tumors with specific FA defects to chemotherapeutic agents.

XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair.

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Abstract 3196Poster Board III-133The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . DisclosuresNo relevant conflicts of interest to declare.

Oxidative-Stress Specific Interaction Between FANCD2 and FOXO3a.

Abstract 1089Poster Board I-111Fanconi anemia (FA) is a human genomic instability syndrome that is uniquely sensitive to oxidative stress. Members of the FA protein family are involved in repair of genetic damage caused by DNA cross-linkers. The molecular pathway in which the FA proteins function in oxidative stress response has not been defined. Here we report functional interaction between the FA protein FANCD2 and the forkhead transcription factor FOXO3a in response to oxidative stress. FOXO3a was colocalized to FANCD2 foci in cells subjected to oxidative stress. The FANCD2-FOXO3a complex was not detected in cells deficient for the FA core complex component FANCA, but could be restored after complementation with a functional FANCA. Consistent with this, a non-monoubiquitinated FANCD2 mutant failed to bind FOXO3a. While both DNA cross-linker mitomycin C and ionizing radiation induced monoubiquitination of FANCD2, neither was able to induce the association of FANCD2 and FOXO3a. This indicates that the FOXO3a-FANCD2 interaction is oxidative stress specific. Overexpression of FOXO3a reduced abnormal accumulation of reactive oxygen species, enhanced cellular resistance to oxidative stress, and increased antioxidant gene expression in corrected but not mutant FA-D2 cells. The novel oxidative stress response pathway converging FANCD2 and FOXO3a identified in this study is likely to contribute to cellular anti-oxidant defense. DisclosuresNo relevant conflicts of interest to declare.

Utility of DNA Repair Protein Foci for the Detection of Putative BRCA1 Pathway Defects in Breast Cancer Biopsies

The DNA damage response pathway controlled by the breast cancer and Fanconi anemia (FA) genes can be disrupted by genetic or epigenetic mechanisms in breast cancer. Defects in this pathway may render the affected tumors hypersensitive to DNA-damaging agents. The identification of these defects poses a challenge because of the large number of genes involved in the FA/BRCA pathway. Many pathway components form subnuclear repair protein foci upon exposure to ionizing radiation in vitro, but it was unknown whether foci can be detected in live cancer tissues. Thus, the goal of this pilot study was to identify pathway defects by using a novel ex vivo foci biomarker assay on tumor biopsies. Fresh pretreatment biopsy specimens from patients with locally advanced sporadic breast cancer were irradiated or mock-treated in the laboratory (ex vivo). Foci formation of DNA repair proteins BRCA1, FANCD2, and RAD51 was detected by immunofluorescence microscopy. Three out of seven tumors showed intact radiation-induced foci formation, whereas the other four tumors exhibited a defective foci response. Notably, three of the foci-defective tumors were estrogen receptor/progesterone receptor/HER2-negative (triple-negative), a phenotype that has been associated with BRCA1 deficiency. In conclusion, in this pilot study, we report the successful detection of BRCA1, FANCD2, and RAD51 foci in breast cancer biopsies irradiated ex vivo. Our approach represents a potentially powerful biomarker assay for the detection of pre-existing and functionally important defects within the complex FA/BRCA pathway, which may ultimately allow us to tailor cancer treatment to the DNA repair profile of individual tumors.

MRE11–RAD50–NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M

AbstractFANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a “clean” FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM−/− cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM−/− cells were sensitive to UV light. FANCM and a C-terminal deletion mutant rescued the cross-linker sensitivity of FANCM−/− cells, whereas a FANCM ATPase mutant did not. Because both mutants restored the formation of FANCD2 foci, we conclude that FANCM functions in an FA core complex–dependent and –independent manner.

Inactivation of Murine Usp1 Results in Genomic Instability and a Fanconi Anemia Phenotype

Fanconi anemia (FA) is a human genetic disease characterized by chromosome instability, cancer predisposition, and cellular hypersensitivity to DNA crosslinking agents. The FA pathway regulates the repair of DNA crosslinks. A critical step in this pathway is the monoubiquitination and deubiquitination of FANCD2. Deubiquitination of FANCD2 is mediated by the ubiquitin protease, USP1. Here, we demonstrate that targeted deletion of mouse Usp1 results in elevated perinatal lethality, male infertility, crosslinker hypersensitivity, and an FA phenotype. Usp1(-/-) mouse embryonic fibroblasts had heightened levels of monoubiquitinated Fancd2 in chromatin. Usp1(-/-) cells exhibited impaired Fancd2 foci assembly and a defect in homologous recombination repair. Double knockout of Usp1 and Fancd2 resulted in a more severe phenotype than either single knockout. Our results indicate that mouse Usp1 functions downstream in the FA pathway. Deubiquitination is a critical event required for Fancd2 nuclear foci assembly, release from chromatin, and function in DNA repair.

A novel cell‐free screen identifies a potent inhibitor of the Fanconi anemia pathway

The Fanconi Anemia (FA) DNA damage response pathway is involved in the processing of DNA interstrand crosslinks (ICLs). As such, inhibition of the FA pathway could chemosensitize FA-competent tumor cells to commonly used ICL agents like cisplatin. Moreover, suppression of the FA pathway is synthetic lethal with deficiencies in several other DNA repair pathways, suggesting that FA pathway inhibitors could be used in targeted therapies against specific tumors. To identify such inhibitors, we designed a novel in vitro screening assay utilizing Xenopus egg extracts. Using the DNA-stimulated monoubiquitylation of Xenopus FANCD2 (xFANCD2-L) as readout, a chemical library screen identified DDN (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone) as a novel and potent FA pathway inhibitor. DDN inhibited xFANCD2-L formation in a dose-dependent manner in both extracts and human cells without disruption of the upstream FA core complex. DDN also inhibited the characteristic subnuclear FANCD2 foci formation following DNA damage. Moreover, DDN displayed a greater synergistic effect with cisplatin in a FA-proficient cancer cell line compared to its FA-deficient isogenic counterpart, suggesting that DDN might be a good lead candidate as cisplatin chemosensitizer in both FA-deficient and FA-competent tumors. This system constitutes the first cell-free screening assay for identifying compounds that inhibit the FA pathway and provides a new biochemical platform for mapping the functions of its various components with specific chemical inhibitors.

A Novel Cell-Free Assay Identifies the Curcumin Analog EF24 as a Potent Inhibitor of the Fanconi Anemia Pathway

Objective: The Fanconi anemia (FA) pathway is a DNA damage response network involved in the cellular resistance against DNA interstrand crosslinks (ICLs). A recent study showed that the FA pathway is synthetic lethal with several other DNA repair genes (Kennedy, 2007) such as ATM, NBS1, RAD54B and TP53BP1. Defects in those genes have been linked to a wide range of inherited and sporadic hematological malignancies including B-CLL, ALL, AML, CML, non-Hodgkin lymphoma, mantle cell lymphoma and multiple myeloma. FA pathway inhibitors may therefore selectively kill malignant cells bearing these defects. Curcumin, a natural product, was the first identified FA pathway inhibitor with activity in the micromolar range in cells (Chirnomas, 2006). However, the poor bioavailability of curcumin hinders its clinical efficacy. Identification of a curcumin analog with better activity, bioavailability and low toxicity could overcome this obstacle. We recently developed a cell-free assay for FA pathway function using Xenopus egg extracts to test the activity of curcumin analogs. As a pilot study we evaluated how well the assay identified inhibitors of the FA pathway in human cells.Methods: Fourteen curcumin analogs previously assayed in the NCI anticancer cell line screen (Adams, 2004) were tested for their activity on the FA pathway. Xenopus egg extracts were used to measure the relative inhibitory activity of the analogs on FANCD2 monoubiquitylation (FANCD2-L) and phosphorylation of other DNA damage response proteins. The underlying mechanism of inhibition was explored by testing the integrity of the core complex, the recruitment of the core complex to DNA and chromatin, and analyzing DNA replication and proteasome activity. Activity of several analogs was confirmed in HeLa cells by evaluation of the inhibition of hydroxyurea (HU)-induced FANCD2-L and FANCD2 foci.Results: EF24 (Adams, 2005) and three structurally similar analogs were 10 times more active than curcumin for FANCD2-L inhibition in Xenopus extracts. These analogs inhibited Mre11 phosphorylation at similar concentrations but had no effect on RPA32 and H2AX phosphorylation. In contrast to curcumin, EF24 did not display significant proteasome inhibition activity and did not affect integrity of the core complex or its recruitment to DNA and chromatin, ruling out these mechanisms to explain inhibition of the FA pathway. In HU-treated HeLa cells, EF24 strongly inhibited FANCD2-L and FANCD2 foci with an IC50 of 350 nM, confirming the results observed in Xenopus extracts.Conclusions: EF24 is a more potent FA pathway inhibitor than curcumin both in Xenopus extracts and in human cells, and as such may be effective as a single agent in targeted therapies against hematological malignancies deficient in ATM, NBS1, RAD54B or TP53BP1. In addition, this study demonstrates that Xenopus extracts are a powerful tool to identify and evaluate small molecules that modulate the FA and other DNA damage response pathways.

Fanconi Anemia and the Response of Human Hematopoietic Cells to DNA Damage

Given their critical role in the generation and renewal of the hematopoietic system, we hypothesize that hematopoietic stem and primitive cells employ unusually stringent DNA damage responses to maintain genome stability. We also propose that Fanconi anemia proteins prevent bone marrow failure through their participation in these DNA repair and damage response pathways. We are testing both hypotheses by characterizing cellular responses to DNA double-strand breaks (DSBs) in 3 lineage-negative populations of hematopoietic cells isolated from human umbilical cord blood: hematopoietic stem cell-containing primitive CD34+ CD38− CD45RA−, early progenitor CD34+ CD38+ and late progenitor CD34− CD38+.Two key events in the initial cellular response to DSBs are phosphorylation of H2AX histones in chromatin at or near DSBs, and association of 53BP1 with gamma-H2AX at DSB sites. Once DSBs are detected, they then are repaired by two major pathways: error-prone non-homologous end-joining (NHEJ), utilized throughout the cell cycle, and error-free homologous recombination (HR), used when sister chromatids are present during S/G2 phases of the cell cycle.To study the response to and repair of DSBs in our sorted hematopoietic populations, we exposed quiescent and cytokine-stimulated haematopoietic cells and human fibroblasts to ionizing radiation (IR). Irradiation with 3 Gy rapidly induced formation of 53BP1 foci in all cell types, both cycling and non-cycling. In contrast, X-irradiation only induced foci of the Fanconi anemia protein FANCD2 in cycling cells. The majority of the induced FANCD2 foci colocalized with foci of gamma-H2AX.The subsequent persistence of 53BP1 foci at X-ray induced damage sites differed significantly between the lin− hematopoietic cells and the fibroblasts. During the first 6 hours following IR, cycling primitive and progenitor hematopoietic cells resolved 53BP1 foci more slowly than cycling human primary fibroblasts. In addition, X-ray induced 53BP1 foci persisted even longer in quiescent primitive CD34+ CD38− and early progenitor CD34+ CD38+ cells than in their cycling counterparts, while foci resolution kinetics were similar in quiescent and cycling late progenitor CD34− CD38+ cells and in fibroblasts. We also used a neutral comet assay to determine the kinetics of DSB rejoining following exposure to 15 Gy. We found that resolution of 53BP1 foci is not directly linked to rejoining of DSBs in lin− hematopoietic cells, as they rejoin DSBs more rapidly than primary fibroblasts, yet resolve 53BP1 foci more slowly. The effect is most prominent in primitive CD34− CD38− cells. Both cycling and non-cycling fibroblasts displayed similar kinetics of DSB rejoining. However, quiescent lin− hematopoietic cells displayed persistent breaks compared to cycling counterparts or to fibroblasts. Of particular note, quiescent lin− hematopoietic cells still had not rejoined a significant fraction of induced DSBs 24 hours post irradiation.Our results indicate that human primitive and progenitor hematopoetic cells differ from fibroblasts in their DNA damage responses and DSB repair kinetics. Our findings also suggest that, unlike fibroblasts, progression through the cell cycle plays a key role in the rapid repair of DSBs in hematopoietic cells. Because Lin− hematopoietic cells are more efficient at rejoining DSBs when cycling, we suggest that error-free HR repair plays an important role in DSB repair in these cells. Our detection of DNA damage-induced FANCD2 foci only in cycling lin− hematopoietic cells provides indirect support for this conclusion, as FA proteins are thought to be involved in HR repair. We are currently testing this hypothesis by determining the effect of knocking down FA protein expression on DSB repair and DNA damage responses in our three hematopoietic populations.

HES1 is a novel interactor of the Fanconi anemia core complex

AbstractFanconi anemia (FA) proteins are thought to play a role in chromosome stability and repair of DNA cross-links; however, these functions may not fully explain the developmental abnormalities and bone marrow failure that are characteristic of FA individuals. Here we associate the FA proteins with the Notch1 developmental pathway through a direct protein-protein interaction between the FA core complex and the hairy enhancer of split 1 (HES1). HES1 interaction with FA core complex members is dependent on a functional FA pathway. Cells depleted of HES1 exhibit an FA-like phenotype that includes cellular hypersensitivity to mitomycin C (MMC) and lack of FANCD2 monoubiquitination and foci formation. HES1 is also required for proper nuclear localization or stability of some members of the core complex. Our results suggest that HES1 is a novel interacting protein of the FA core complex.

ERCC1 is required for FANCD2 focus formation

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by γH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.

Therapeutic Exploitation of Tumor Cell Defects in Homologous Recombination

In the decade since the BRCA1 and BRCA2 genes were cloned, much has been learned about the function of these two major causes of familial breast cancer. BRCA2 has been shown to play a direct role in the repair of DNA by homologous recombination, by interacting with the Rad51 protein and facilitating the formation of Rad51 aggregates at the site of DNA damage. It likely plays a similar role when double strand breaks are created in the course of normal DNA replication; the absence of BRCA2 results in chromosomal instability, which is likely secondary to the defect in DNA repair. In the absence of BRCA2, the cell is more dependent on residual repair via Rad52, which makes Rad52 a target for therapy in BRCA-deficient tumors. BRCA1 plays a role in sensing DNA damage and replication stress and mediating the signaling responses. Therefore, in addition to its role in mediating DNA repair by homologous recombination via BRCA2, it can also signal cell cycle checkpoints and mediate other transcriptional responses to DNA damage. We have argued that the mechanism of cancer susceptibility from BRCA1 or BRCA2 deficiency is mediated via the defect in homologous recombination, since it is the main feature they share in common. We and others have recently demonstrated that the defect in homologous recombination changes the drug sensitivity profile, rendering the BRCA-deficient breast cancers sensitive to MitomycinC, cisplatin, etoposide and other drugs that produce complex double-stranded lesions in DNA. Furthermore, they show resistance to taxanes and navelbine. Fanconi anemia defective cells also show sensitivity to the same class of drugs, although their defect in homologous recombination in response to strand breaks appears less marked than in BRCA-deficient cells. However, Fanconi anemia cells also show chromosomal fragility, and appear to have defects in maintenance of the replication fork. Therefore, knowledge of whether this specific DNA repair pathway of homologous recombination is defective in breast cancer cells would be valuable information in planning optimized individual therapy. We have developed techniques to measure the functional integrity of homologous recombination in human breast cancers. Core biopsy samples are obtained and immediately irradiated ex vivo, allowing 3-4 hours for the appearance of Rad51, BRCA1 and FancD2 foci. Thin sections are obtained, permeabilized and stained by immunofluorescent techniques. We have identified tumors with defects in the ability to form Rad51 and BRCA1 foci, where there is no known genetic predisposition, implying that this BRCA-dependent repair pathway may be inactivated in sporadic as well as familial breast cancers. Thus, functional assays of homologous recombination could become a useful technique to determine phenotype of human breast cancer, which in turn will influence the choice of therapy.

Biomarkers and Mechanisms of FANCD2 Function

Genetic or epigenetic inactivation of the pathway formed by the Fanconi anemia (FA) and BRCA1 proteins occurs in several cancer types, making the affected tumors potentially hypersensitive to DNA cross-linkers and other chemotherapeutic agents. It has been proposed that the inability of FA/BRCA-defective cells to form subnuclear foci of effector proteins, such as FANCD2, can be used as a biomarker to aid individualization of chemotherapy. We show that FANCD2 inactivation not only renders cells sensitive to cross-links, but also oxidative stress, a common effect of cancer therapeutics. Oxidative stress sensitivity does not correlate with FANCD2 or RAD51 foci formation, but associates with increased γH2AX foci levels and apoptosis. Therefore, FANCD2 may protect cells against cross-links and oxidative stress through distinct mechanisms, consistent with the growing notion that the pathway is not linear. Our data emphasize the need for multiple biomarkers, such as γH2AX, FANCD2, and RAD51, to capture all pathway activities.

Human Mus81 and FANCB independently contribute to repair of DNA damage during replication

Recent studies suggest a crucial role for homologous recombination (HR) in repairing replication-associated DNA lesions. In mammals, the Mus81 endonuclease and the Fanconi anemia (FA) pathway have been implicated in HR repair; however, their functional relationship has remained unexplored. Here, we knockout the genes for Mus81 and FANCB, a component of the FA core complex, in the human Nalm-6 cell line. We show that Mus81 plays an important role in cell proliferation to suppress cell death when FANCB is missing, indicating a functional linkage between Mus81 and the FA pathway. In DNA cross-link repair, roles for Mus81 and the FA pathway appear to have an overlapping function. Intriguingly, Mus81 and FANCB act independently in surviving exposure to camptothecin (CPT). Although CPT-induced FANCD2 and Mus81 foci co-localize with Rad51, loss of Mus81, but not FANCB, results in significantly decreased levels of spontaneous and CPT-induced sister chromatid exchanges (SCEs). In addition, Mus81, unlike FANCB, has no significant role in gene targeting as well as in repairing hydroxyurea (HU)-induced stalls of replication forks. Collectively, our results provide the first evidence for differential functions of Mus81 and the FA pathway in repair of DNA damage during replication in human cells.

The Human Papillomavirus Type 16 E7 Oncoprotein Activates the Fanconi Anemia (FA) Pathway and Causes Accelerated Chromosomal Instability in FA Cells

Fanconi anemia (FA) patients have an increased risk for squamous cell carcinomas (SCCs) at sites of predilection for infection with high-risk human papillomavirus (HPV) types, including the oral cavity and the anogenital tract. We show here that activation of the FA pathway is a frequent event in cervical SCCs. We found that FA pathway activation is triggered mainly by the HPV type 16 (HPV-16) E7 oncoprotein and is associated with an enhanced formation of large FANCD2 foci and recruitment of FANCD2 as well as FANCD1/BRCA2 to chromatin. Episomal expression of HPV-16 oncoproteins was sufficient to activate the FA pathway. Importantly, the expression of HPV-16 E7 in FA-deficient cells led to accelerated chromosomal instability. Taken together, our findings establish the FA pathway as an early host cell response to high-risk HPV infection and may help to explain the greatly enhanced susceptibility of FA patients to squamous cell carcinogenesis at anatomic sites that are frequently infected by high-risk HPVs.

Proteasome Function Is Required for DNA Damage Response and Fanconi Anemia Pathway Activation

Proteasome inhibitors sensitize tumor cells to DNA-damaging agents, including ionizing radiation (IR), and DNA cross-linking agents (melphalan and cisplatin) through unknown mechanisms. The Fanconi anemia pathway is a DNA damage-activated signaling pathway, which regulates cellular resistance to DNA cross-linking agents. Monoubiquitination and nuclear foci formation of FANCD2 are critical steps of the Fanconi anemia pathway. Here, we show that proteasome function is required for the activation of the Fanconi anemia pathway and for DNA damage signaling. Proteasome inhibitors (bortezomib and MG132) and depletion of 19S and 20S proteasome subunits (PSMD4, PSMD14, and PSMB3) inhibited monoubiquitination and/or nuclear foci formation of FANCD2, whereas depletion of DSS1/SHFM1, a subunit of the 19S proteasome that also directly binds to BRCA2, did not inhibit FANCD2 monoubiquitination or foci formation. On the other hand, DNA damage-signaling processes, such as IR-induced foci formation of phosphorylated ATM (phospho-ATM), 53BP1, NBS1, BRCA1, FANCD2, and RAD51, were delayed in the presence of proteasome inhibitors, whereas ATM autophosphorylation and nuclear foci formation of gammaH2AX, MDC1, and RPA were not inhibited. Furthermore, persistence of DNA damage and abrogation of the IR-induced G(1)-S checkpoint resulted from proteasome inhibition. In summary, we showed that the proteasome function is required for monoubiquitination of FANCD2, foci formation of 53BP1, phospho-ATM, NBS1, BRCA1, FANCD2, and RAD51. The dependence of specific DNA damage-signaling steps on the proteasome may explain the sensitization of tumor cells to DNA-damaging chemotherapeutic agents by proteasome inhibitors.

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.

A requirement of FancL and FancD2 monoubiquitination in DNA repair

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