Fanconi Anemia Group Research Articles

Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein (FANCJ) Patient Mutations

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding and remodeling of structured DNA or RNA, which is coordinated by conserved helicase motifs. FANCJ is a DNA helicase that is genetically linked to Fanconi anemia, breast cancer, and ovarian cancer. Here, we characterized two Fanconi anemia patient mutations, R251C and Q255H, that are localized in helicase motif Ia. Our genetic complementation analysis revealed that both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, our biochemical assays demonstrated that both purified recombinant proteins abolished DNA helicase activity and failed to disrupt the DNA-protein complex. Intriguingly, R251C impaired DNA binding ability to single-strand DNA and double-strand DNA, whereas Q255H retained higher binding activity to these DNA substrates compared with wild-type FANCJ protein. Consequently, R251C abolished its DNA-dependent ATP hydrolysis activity, whereas Q255H retained normal ATPase activity. Physically, R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA. Although both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. Taken together, our work not only reveals the structural function of helicase motif Ia but also provides the molecular pathology of FANCJ in related diseases.

The Fanconi Anemia Group C Protein Interacts with Uncoordinated 5A and Delays Apoptosis

The Fanconi anemia group C protein (FANCC) is one of the several proteins that comprise the Fanconi anemia (FA) network involved in genomic surveillance. FANCC is mainly cytoplasmic and has many functions, including apoptosis suppression through caspase-mediated proteolytic processing. Here, we examined the role of FANCC proteolytic fragments by identifying their binding partners. We performed a yeast two-hybrid screen with caspase-mediated FANCC cleavage products and identified the dependence receptor uncoordinated-5A (UNC5A) protein. Here, we show that FANCC physically interacts with UNC5A, a pro-apoptotic dependence receptor. FANCC interaction occurs through the UNC5A intracellular domain, specifically via its death domain. FANCC modulates cell sensitivity to UNC5A-mediated apoptosis; we observed reduced UNC5A-mediated apoptosis in the presence of FANCC and increased apoptosis in FANCC-depleted cells. Our results show that FANCC interferes with UNC5A's functions in apoptosis and suggest that FANCC may participate in developmental processes through association with the dependence receptor UNC5A.

Basal level of FANCD2 monoubiquitination is required for the maintenance of a sufficient number of licensed-replication origins to fire at a normal rate

Normal DNA replication starts following the stepwise recruitment of replication initiators to assemble Mini-chromosome Maintenance (MCM) 2-7 protein complexes at an adequate amount of DNA replication origins. Under normal conditions, the monoubiquitination of Fanconi Anemia (FA) group D2 protein (FANCD2) occurs in each S-phase of cell cycle, which is the basal level of FANCD2 monoubiquitination. However, little is known regarding the roles of this basal level of monoubiquitinated FANCD2. Here we show that monoubiquitinated FANCD2 in each S-phase of normal cell cycle is essential for replication origins to fire at a normal rate. We found that the basal level of the monoubiquitinated FANCD2 can interact with replication origins as well as mini-chromosome maintenance protein 3 (MCM3) in an S-phase specific manner to secure an enough number of the licensed-origins to fire. Non-monoubiquitinated FANCD2 or mutant MCM3 lacking AA 477-480 responsible for interacting with FANCD2 can lead to an insufficient amount of licensed origins to fire and, thereby, enlarged intervals between the fired origins. Our results demonstrate that the monoubiquitinated FANCD2 in each S-phase of normal cell cycle is required to maintain an enough number of licensed origins to initiate the normal DNA replication. This finding is the first to provide insights into how FANCD2 functions under normal condition of cell cycle to maintain genome stability, as well as resulting implications in the strategic improvement for the fight against human cancer.

Chromosomal instability in patients with Fanconi anemia from Serbia.

Fanconi anemia (FA) is a rare hereditary disease in a heterogeneous group of syndromes, so-called chromosome breakage disorders. Specific hypersensitivity of its cells to chemical agents, such as diepoxybutane (DEB), was used as a part of screening among patients with clinical suspicion of FA. The aim of this study was to determine chromosomal instability in patients with FA symptoms in Serbia. A total of 70 patients with phenotypic symptoms of FA, diagnosed at the Mother and Child Health Care Institute of Serbia "Dr Vukan Cupić, Belgrade and University Children's Hospital, Belgrade from February 2004 to September 2011, were included in this study. Cytogenetic instability analysis was performed on untreated and DEB-treated 72 h-cultures of peripheral blood. Ten patients in the group of 70 suspected of FA, showed increased DEB induced chromosome breakage and were classified into the FA group. The range of DEB induced aberrant cells percentages in the FA group was from 32% to 82%. DEB sensitivity of 58 tested patients were bellow FA values (range: 0-6%) (non-FA group), with no overlapping. The remaining two patients showed borderline sensitivity (borderline FA group - FA*), comparing to the healthy controls. This study revealed 10 patients with FA on the basis of cytogenetic analysis of DEB induced chromosome aberrations. Our results are in consistency with those from the literature. Early and precise diagnosis of FA is very important in further treatment of these patients, considering its cancer prone and lethal effects.

Elevated levels of STAT1 in Fanconi anemia group A lymphoblasts correlate with the cells' sensitivity to DNA interstrand crosslinking drugs

Progressive bone marrow failure starting in the first decade of life is one of the main characteristics of Fanconi anemia. Along with the bone marrow failure, this pathology is characterized by congenital malformations, endocrine dysfunction and an extraordinary predisposition to develop cancer. The fact that hematopoietic progenitor cells from subjects with Fanconi anemia are sensitive to both DNA-interstrand crosslinking agents and inflammatory cytokines, which are aberrantly overproduced in these patients, has led to different explanations for the causes of the bone marrow failure. We analyzed STAT1 expression in lymphoblastoid cell lines derived from patients with Fanconi anemia group A and correlated this with aspects of the Fanconi anemia phenotype such as sensitivity to genotoxic agents or to inhibitory cytokines. We provide evidence of overexpression of STAT1 in FANCA-deficient cells which has both transcriptional and post-translational components, and is related to the constitutive activation of ERK in Fanconi anemia group A cells, since it can be reverted by treatment with U0126. STAT1 phosphorylation was not defective in the lymphoblasts, so these cells accumulated higher levels of active STAT1 in response to interferon gamma, probably in relation to their greater sensitivity to this cytokine. On the other hand, inhibition of STAT1 by genetic or chemical means reverted the hypersensitivity of Fanconi anemia group A lymphoblasts to DNA interstrand crosslinking agents. Our data provide an explanation for the mixed sensitivity of Fanconi anemia group A cells to both genotoxic stress and inflammatory cytokines and indicate new targets for the treatment of bone marrow failure in these patients.

Fanconi anemia proteins interact with CtBP1 and modulate the expression of the Wnt antagonist Dickkopf-1

AbstractFanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure, and increased susceptibility to cancer. Of the fifteen FA proteins, Fanconi anemia group C (FANCC) is one of eight FA core complex components of the FA pathway. Unlike other FA core complex proteins, FANCC is mainly localized in the cytoplasm, where it is thought to function in apoptosis, redox regulation, cytokine signaling, and other processes. Previously, we showed that regulation of FANCC involved proteolytic processing during apoptosis. To elucidate the biological significance of this proteolytic modification, we searched for molecular interacting partners of proteolytic FANCC fragments. Among the candidates obtained, the transcriptional corepressor protein C-terminal binding protein-1 (CtBP1) interacted directly with FANCC and other FA core complex proteins. Although not required for stability of the FA core complex or ubiquitin ligase activity, CtBP1 is essential for proliferation, cell survival, and maintenance of chromosomal integrity. Expression profiling of CtBP1-depleted and FA-depleted cells revealed that several genes were commonly up- and down-regulated, including the Wnt antagonist Dickkopf-1 (DKK1). These findings suggest that FA and Wnt signaling via CtBP1 could share common effectors.

BIK (NBK) is a mediator of the sensitivity of Fanconi anaemia group C lymphoblastoid cell lines to interstrand DNA cross-linking agents

FA (Fanconi anaemia) is a rare hereditary disorder characterized by congenital malformations, progressive bone marrow failure and an extraordinary predisposition to develop cancer. At present, 15 genes have been related to this condition and mutations of them have also been found in different types of cancer. Bone marrow failure threatens the life of FA patients during the first decade of their life, but the mechanisms underlying this process are not completely understood. In the present study we investigate a possible imbalance between the expression of pro- and anti-apoptotic proteins as a cause for the hypersensitivity of FANCC (FA, complementation group C)-deficient cells to genotoxic stress. We found a BIK (Bcl-2 interacting killer) over-expression in lymphoblastoid cell lines derived from FA-C patients when compared with their phenotypically corrected counterparts. This overexpression has a transcriptional basis since the regulatory region of the gene shows higher activity in FANCC-deficient cells. We demonstrate the involvement of BIK in the sensitivity of FA-C lymphoblasts to interstrand DNA cross-linking agents as it is induced by these drugs and interference of its expression in these cells preserves their viability and reduces apoptosis. We investigate the mechanism of BIK overexpression in FANCC-deficient cells by analysing the activity of many different signalling pathways in these cells. Finally, we provide evidence of a previously undescribed indirect epigenetic regulation of BIK in FA-C lymphoblasts mediated by ΔNp73, an isoform of p73 lacking its transactivation domain that activates BIK through a proximal element in its promoter.

The antitumor agent doxorubicin binds to Fanconi anemia group F protein

Doxorubicin, a commonly used cancer chemotherapy agent, elicits several potent biological effects, including synergistic-antitumor activity in combination with cisplatin. However, the mechanism of this synergism remains obscure. Here, we employed an improved T7 phage display screening method to identify Fanconi anemia group F protein (FANCF) as a doxorubicin-binding protein. The FANCF-doxorubicin interaction was confirmed by pull-down assay and SPR analysis. FANCF is a component of the Fanconi anemia complex, which monoubiquitinates D2 protein of Fanconi anemia group as a cellular response against DNA cross-linkers such as cisplatin. We observed that the monoubiquitination was inhibited by doxorubicin treatment.

Inhibition of ATR-Dependent Signaling by Protoapigenone and Its Derivative Sensitizes Cancer Cells to Interstrand Cross-link–Generating Agents In Vitro and In Vivo

DNA damage caused during cancer treatment can rapidly activate the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR)-dependent phosphorylation of Chk2 and Chk1 kinases, which are hallmarks of the DNA damage response (DDR). Pharmacologic inhibition of ATR causes a synthetic lethal effect on ATM- or p53-defective cancers, suggesting that such inhibition is an effective way to improve the sensitivity of cancers to DNA-damaging agents. Here, both the natural compound protoapigenone (WYC02) and its synthetic derivative WYC0209 exhibited cytotoxic effects on various cancer cell lines. WYC02 causes chromosomal aberration in the mitotic spreads of Chinese hamster ovary cells. Interestingly, cancer cells did not exhibit typical DDR markers upon exposure to WYC02 and WYC0209 (WYCs). Further investigation into the molecular mechanisms of WYCs function revealed that they have a potential ability to inhibit DDR, particularly on activation of Chk1 and Fanconi anemia group D2 protein (FANCD2), but not Chk2. In this way, WYCs inhibited ATR-mediated DNA damage checkpoint and repair. Furthermore, when combined with the DNA cross-linking agent cisplatin, treatment with WYCs resulted in increased tumor sensitivity to interstrand cross-link-generating agents both in vitro and in vivo. Our results therefore especially implicate WYCs in enhancing tumor chemosensitivity when the ATR checkpoint is constitutively active in states of oncogene-driven replicative stress or tolerance to DNA-interfering agents.

The Q Motif of Fanconi Anemia Group J Protein (FANCJ) DNA Helicase Regulates Its Dimerization, DNA Binding, and DNA Repair Function

The Q motif, conserved in a number of RNA and DNA helicases, is proposed to be important for ATP binding based on structural data, but its precise biochemical functions are less certain. FANCJ encodes a Q motif DEAH box DNA helicase implicated in Fanconi anemia and breast cancer. A Q25A mutation of the invariant glutamine in the Q motif abolished its ability to complement cisplatin or telomestatin sensitivity of a fancj null cell line and exerted a dominant negative effect. Biochemical characterization of the purified recombinant FANCJ-Q25A protein showed that the mutation disabled FANCJ helicase activity and the ability to disrupt protein-DNA interactions. FANCJ-Q25A showed impaired DNA binding and ATPase activity but displayed ATP binding and temperature-induced unfolding transition similar to FANCJ-WT. Size exclusion chromatography and sedimentation velocity analyses revealed that FANCJ-WT existed as molecular weight species corresponding to a monomer and a dimer, and the dimeric form displayed a higher specific activity for ATPase and helicase, as well as greater DNA binding. In contrast, FANCJ-Q25A existed only as a monomer, devoid of helicase activity. Thus, the Q motif is essential for FANCJ enzymatic activity in vitro and DNA repair function in vivo.

Incidence of liver abnormalities in Fanconi anemia patients

Patients with Fanconi anemia (FA) are prone to liver tumors, especially after androgen treatment, but other liver abnormalities have not been described for these patients. Here, we systematically reviewed liver manifestations in a cohort of 64 adult and pediatric patients with FA followed in a single center. "Significant biological liver abnormalities(SBLA)" in the absence of any androgen treatment were found in five patients, including two children, belonging to rare FA groups; these two patients presented with a very severe chronic cytolysis pattern which may be classified as a new FA phenotype. Liver radiological abnormalities, which include hepatic tumors (n 5 4), hepatomegaly(n 5 1), hyperechogenicity (n 5 2), and a previously undescribed biliary duct dilatation as demonstrated by magnetic resonance cholangiopancreatography(MRCP) (n 5 2), were found in eight patients who received androgen treatment or who had iron overload. Lastly, we found no correlation between cytolysis intensity and high levels of alpha-fetoprotein (AFP); this common finding in FA patients cannot therefore be explained by hepatocyte regeneration.

Physical and Functional Interaction Between Fanconi Anemia Group J Helicase and MRE11 Nuclease

FANCJ mutations are linked to the chromosomal instability disorder Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double‐strand breaks (DSBs) and interstrand cross‐links (ICLs), but its mechanism of action is not well understood. Live cell imaging demonstrates that FANCJ recruitment to laser‐induced DSBs, but not psoralen‐ICLs, is dependent on nuclease‐active MRE11 and independent of BRCA1. Inhibition of MRE11 nuclease by Mirin adversely affected FANCJ localization to laser‐induced DSBs, but not psoralen‐ICLs. FANCJ is required for timely recruitment of Bloom's syndrome helicase and CtIP to DSBs in vivo. FANCJ directly interacts with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ harnesses MRE11 nuclease to facilitate DSB processing and appropriate end resection. Spontaneous or IR‐induced chromosomal instability in FANCJ‐depleted cells is suppressed when MRE11 is depleted. Interplay between FANCJ and MRE11 insures a normal response to IR‐induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and FANCA, but not FANCD2. Thus, MRE11 helps to recruit FANCJ to DSBs and FANCJ can modulate MRE11 nuclease activity at DNA ends, consistent with a collaborative role of FANCJ and BLM helicases with MRE11 in strand resection.

BRCA1-mediated repression of mutagenic end-joining of DNA double-strand breaks requires complex formation with BACH1

BACH1 (BRCA1-associated C-terminal helicase 1), the product of the BRIP1 {BRCA1 [breast cancer 1, early onset]-interacting protein C-terminal helicase 1; also known as FANCJ [FA-J (Fanconi anaemia group J) protein]} gene mutated in Fanconi anaemia patients from complementation group J, has been implicated in DNA repair and damage signalling. BACH1 exerts DNA helicase activities and physically interacts with BRCA1 and MLH1 (mutL homologue 1), which differentially control DNA DSB (double-strand break) repair processes. The present study shows that BACH1 plays a role in both HR (homologous recombination) and MMEJ (microhomology-mediated non-homologous end-joining) and reveals discrete mechanisms underlying modulation of these pathways. Our results indicate that BACH1 stimulates HR, which depends on the integrity of the helicase domain. Disruption of the BRCA1-BACH1 complex through mutation of BACH1 compromised errorfree NHEJ (non-homologous end-joining) and accelerated error-prone MMEJ. Conversely, molecular changes in BACH1 abrogating MLH1 binding interfered neither with HR nor with MMEJ. Importantly, MMEJ is a mutagenic DSB repair pathway, which is derepressed in hereditary breast and ovarian carcinomas. Since BRCA1 and BACH1 mutations targeting the BRCA1-BACH1 interaction have been associated with breast cancer susceptibility, the results of the present study thus provide evidence for a novel role of BACH1 in tumour suppression.

Immunological Prophile of FA. A Multicentric retrospective Analysis of 61 Patients

Abstract 1347 INTRODUCTION:Fanconi Anemia (FA) is an autosomal and X-linked recessive disease characterized by marrow failure, somatic malformations and cancer proneness primarily leading to AML and head and neck carcinomas. The disease is due to mutations in at least 15 genes responsible for a failure of DNA repair mechanisms that renders the cells sensitive to interstrand cross linkers leading to block of cell cycle in G2 phase. However there is evidence that FA proteins have multiple functions as they are also implicated in cytokine hypersensitivity, response to oxidative stress and immune response. Scanty information is available on immunological status in FA patients. We conducted a retrospective multi-centric analysis of the pre HSCT immunological status of 61 FA patients in aplastic phase by assessing peripheral blood immunophenotype and immunoglobulin serum level (46/61). RESULTS:Absolute lymphocyte number was within the normal range for age in 70% of patients (43/61). Only 10% of subjects (6/61) were severely lymphocytopenic ( <1.0×109/l). Absolute B cells were below the lower limit of the normal range for the age (10th percentile) in 75% of patients. Absolute CD3+ and CD8+ cells were within the normal distribution for age in virtually all cases (98% and 94% respectively) whereas CD4+ cells and NK cells were below the lower normal range in 45% and 49% of cases respectively. Within the T cell population, CD3+CD45RA+ (naive) cells were above the normal range in 68% of patients while CD3+CD45+RO+ (memory) cells were reduced in 60 % patients. In 70% (22/32) of the patients in whom we tested regulatory T cells (CD3+/CD4+/CD25bright+/FoxP3+) they were found below the lower normal range. Serum immunoglobulin A and G were within the age matched range of normal controls whereas IgMs were lower than normal range for age in 50% (23/46) of patients. CONCLUSIONS:This is the largest study ever conducted on the immunological status of FA patients suggesting that these subjects have an impaired immunity which is not directly reflected by the number of circulating lymphocytes. Reduction of B and also of CD4+cells is consistent with low IgM serum levels. Deficiency of regulatory T cells and the relative excess of CD3+and CD8+ lymphocytes are in keeping with the hypothesis that a dysregulated immunity might contribute to the development of marrow failure. In fact it is possible that the lack of inhibitory effect of T regulatory cells might facilitate CD3+ and CD8+ subsets to produce myelosuppressive cytokines TNF-alfa and IFN-gamma to which FA hematopoietic cells are hypersensitive (1–4). The reduction of T memory cells, that constitutionally have a more hampered DNA repair capability vs T naive cells (5), looks consistent with the classical DNA repair deficiency of FA. Overall these data suggest that specific immune function in FA: (a) is impaired in a rather independent fashion from lymphopenia, (b) might contribute to development of marrow failure and (c) might reflect, to some extent, multifunctionality of FA proteins. Next step is to assess cytokine serum levels, ongoing at writing, that may help to further comprehend the above data. -1- Fagerlie SR, Bagby GC. Immune defects in Fanconi anemia. Crit Rev Immunol. 2006;26(1):81–96 -2- Dufour C, Corcione A, Svahn J, Haupt R, Poggi V, Béka’ssy AN, Scimè R, Pistorio, Pistoia V. TNF-alpha and IFN-gamma are overexpressed in the bone marrow of Fanconi anemia patients and TNF-alpha suppresses erythropoiesis in vitro. Blood. 2003 Sep 15;102(6):2053–9. −3 - Briot D, Macé-Aimé G, Subra F, Rosselli F. Aberrant activation of stress-response pathways leads to TNF-alpha oversecretion in Fanconi anemia. Blood. 2008 Feb 15;111(4):1913–23. -4- Vanderwerf SM, Svahn J, Olson S, Rathbun RK, Harrington C, Yates J, Keeble W, Anderson DC, Anur P, Pereira NF, Pilonetto DV, Pasquini R, Bagby GC. TLR8-dependent TNF-(alpha) overexpression in Fanconi anemia group C cells. -5- Scarpaci S, Frasca D, Barattini P, Guidi L, Doria G. DNA damage recognition and repair capacities in human naïve and memory T cells from peripheral blood of young and elderly subjects. Mech Ageing Dev. 2003 Apr;124(4):517–24. Disclosures:No relevant conflicts of interest to declare.

FAD (Fanconi anaemia group D)

Review on FAD (Fanconi anaemia group D), with data on DNA, on the protein encoded, and where the gene is implicated.

Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome

Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ-BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an 'undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.

Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress

Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex-binding compound telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response.

Localization of αII Spectrin to Telomeres After DNA Interstrand Cross-Link Damage Is Deficient In Fanconi Anemia Group A Cells, Which May Lead to Telomere Dysfunction.

AbstractAbstract 1160A hallmark of the bone marrow failure disorder, Fanconi anemia (FA), is genomic instability which is characterized by both chromosome instability and defects in repair of DNA interstrand cross-links (ICLs). The chromosome instability in FA, in particular chromosome end fusions and breaks, suggests that there is telomere dysfunction. We have shown that the structural protein, nonerythroid a spectrin (aIISp), is present in the nucleus of normal human cells where it is critical in both repair of DNA ICLS and maintaining chromosome stability. aIISp is important in recruitment of the ICL repair protein, XPF, to sites of damage. We have shown that there is a deficiency in aIISp in FA cells lines, due to its increased breakdown, which correlates with reduced repair of DNA ICLs and increased chromosomal aberrations after ICL damage. The goal of the present study was to determine whether aIISp plays a role in telomere function after DNA ICL damage and whether telomere dysfunction in FA cells after damage can be corrected when levels of aIISp are restored to normal. Normal human and FA, complementation group A (FA-A) lymphoblastoid cells, which have telomerase, were used. Undamaged cells or cells treated with mitomycin C (MMC), a DNA ICL agent, were studied using immunofluorescence and in situ hybridization (IF-FISH). Cells were probed with anti-a-spectrin and either (C3TA2)3-Cy3 labeled peptide nucleic acid (PNA) telomeric probe or antibodies against two proteins, TRF1 or TRF2, which are components of the shelterin complex that is essential for protecting chromosome ends and preventing telomere dysfunction. The appropriate fluorescent secondary antibodies were used. The results showed that 47–50% of aIISp foci colocalized with the PNA probe, TRF1 or TRF2 in telomeres in MMC damaged but not undamaged normal cells. Levels of colocalization peaked at 16 hours and, by 24 hours, aIISp foci were no longer observed. This colocalization was specific for the S phase of the cell cycle. These results showed that in normal cells aIISp associates with telomeres in S phase after ICL damage and may play an important role in telomere function after damage. After ICLs are presumably unhooked/removed, aIISp dissociates from telomeres. The view that aIISp is involved in telomere function after ICL damage is strengthened by coimmunoprecipitation studies which show that aIISp has markedly enhanced binding for TRF1 and TFR2 after cells were damaged with MMC. IF-FISH also showed that the ICL repair protein, XPF, had enhanced association with telomeres after ICL damage, which further indicated that repair is taking place. In FA-A cells, in which aIISp levels are reduced, aIISp did not colocalize with the PNA probe, with TRF1 or with TRF2 after ICL damage; XPF also did not localize to telomeres after ICL formation. However, after aIISp levels were returned to normal in FA-A cells by siRNA knock down of m-calpain, a protease which cleaves aIISp and levels of which are elevated in FA cells, aIISp as well as XPF associated with telomeres after ICL damage. The chromosomal aberrations (i.e., breaks and end fusions) observed in FA-A cells after ICL damage were also corrected. ICLs can lead to stalled DNA replication forks in S phase. The present results suggest that, after damage, aIISp associates with telomeres in S phase at ICLs at stalled replication forks. Since aIISp is involved in ICL repair, this association could be of particular importance in repair of ICLs in replicating telomeres. In FA-A cells, failure of localization of aIISp and XPF to telomeres after ICL damage could have severe consequences. It could lead to unrepaired ICLs, which in replicating telomeres could lead to telomere dysfunction and to production of the chromosomal aberrations observed in these cells. These studies show that aIISp may play an important role in telomere function after DNA damage and during telomere replication. Correction of the deficiency in aIISp in FA cells by knocking down m-calpain could be significant in reversing telomere dysfunction and chromosomal aberrations after ICL damage and could potentially have important therapeutic relevance. Disclosures:No relevant conflicts of interest to declare.

Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

AbstractFanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.

Fanconi Anemia Group J Mutation Abolishes its DNA Repair Function by Uncoupling DNA Translocation from Helicase Activity

Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and cancer. FANCJ, one of thirteen genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The FANCJ disease‐causing A349P mutation resides immediately adjacent to a highly conserved cysteine of the Iron‐Sulfur domain. Given the clinical severity of the A349P allele, we investigated its effect on the functions of FANCJ. Purified recombinant FANCJ‐A349P protein had reduced iron and was defective in coupling ATP hydrolysis and translocase activity to unwinding forked duplex or G‐quadruplex DNA substrates. The FANCJ‐A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA‐J null cell line as detected by cell survival or ƒ×‐H2AX foci formation. Expression of FANCJ‐A349P in a wild‐type background exerted a dominant negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to transduce force production from ATP hydrolysis to catalytic strand separation or destabilize protein (e.g., RAD51) bound to DNA is required for its role in DNA repair.