Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts

Injection of Fanconi anaemia (complementation group A) fibroblasts with HeLa mRNA is shown to correct their abnormal response to a psoralen cross-linking challenge, namely permanent repression of DNA synthesis. Injection of gradient-fractionated mRNA led to identification of a single fraction, containing mRNA of approximately 650 bases, which is responsible for this effect. This finding suggests that Fanconi anaemia (group A) cells are deficient in a small protein, up to 20 kDa in size, which is involved in the cellular response to DNA interstrand cross-links.     

Arrest of S-phase progression is impaired in Fanconi anemia cells

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.     

Damage-resistant DNA synthesis in Fanconi anemia cells treated with a DNA cross-linking agent

Fanconi anemia (FA) is a recessive disorder associated with diverse congenital anomalies, progressive bone marrow failure, and a marked predisposition to develop cancer. At the cellular level, FA is characterized by a prolonged G(2) phase in proliferating cells and a marked hypersensitivity to both the cytotoxic and the clastogenic effects of agents which produce DNA interstrand cross-links. Treatment with these agents leads to even further prolongation of the G(2) phase in FA cells. We now show that FA cells, from four different complementation groups, fail to decrease their rates of replicative DNA synthesis, as do normal cells, following treatment with a DNA cross-linking agent. This may be responsible for the prolongation of the G2 phase seen in these cells, and suggests that the fundamental defect in response of FA cells to DNA cross-linking agents may be in the S phase, rather than the G(2) phase, of the cell cycle.     

Reduced uptake and incorporation of 3H-thymidine in fanconi anemia fibroblasts

Summary Uptake of ³H-thymidine and its incorporation into DNA was studied in fibroblastic cell lines derived from normal individuals, patients with Fanconi anemia, and those heterozygous for this genetic trait. Uptake and incorporation for the normal cells were about five and seven times higher, respectively, than for Fanconi anemia fibroblasts; mean values for heterozygotes were intermediate. This effect was dependent on the duration of cell exposure to ³H-thymidine and was not observed with other labeled compounds. Thus, a genetically-determined metabolic defect may exist in Fanconi anemia patients which can be readily studied at the cellular level. This finding may be relevant to the observed clinical, cytogenetic, biochemical, and biologic properties related to expression of the Fanconi anemia gene.     

Microinjection of normal cell extracts into Fanconi anemia fibroblasts corrects defective scheduled DNA synthesis recovery after 8-methoxypsoralen plus UVa treatment

Summary Fanconi anemia (FA) cells show an increased sensitivity to 8-methoxypsoralen (8-MOP) plus UVa treatment; after an initial reduction of their semiconservative DNA synthesis rate, they do not recover like normal cells. We microinjected extracts from normal cells into FA fibroblasts from complementation group A and determined semiconservative DNA synthesis rates by autoradiography; the hampered recovery phase was completely restored.The present data are part of M. Gök's thesis 1987 at the University of Heidelberg     

Tip60 is required for DNA interstrand cross-link repair in the Fanconi anemia pathway

The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.     

Light scatter and DNA accessibility to propidium iodide of ataxia telangiectasia and fanconi anemia cells

Cells from individuals with genetic diseases ataxia telangiectasia (AT) and Fanconi anemia (FA) exhibit hypersensitivity to ionizing radiation (AT) or DNA cross-linking agents (FA) which may be caused by multiple factors including defects in chromatin structure and DNA repair. In this study, a combination of cytometric techniques was employed to study the chromatin conformation of AT and FA cells. Nuclei of peripheral blood mononuclear cells (PBMCs) and of skin fibroblasts established from AT and FA patients were analyzed by light scattering and fluorimetric titration with the DNA-intercalating dye propidium iodide. The light scatter measurements revealed the presence of small-sized nuclei with reduced granularity in PBMCs and fibroblasts from both AT and FA patients. The fluorometric titration data could be interpreted by assuming two classes of propidium iodide binding sites with different affinities. The number of high-affinity sites in AT and FA fibroblasts was significantly larger (by 20%) than in control cells. Our findings show the applicability of cytometric techniques for the rapid assessment of chromatin conformation and also suggest the possibility to identify AT and FA carriers.     

Suboptimal action of NF-kappaB in Fanconi anemia cells results from low levels of thioredoxin

Electrophoretic mobility shift assays (EMSA) revealed that under standard cell culture conditions NF-kappaB was induced in Fanconi anemia fibroblasts in contrast to control cells. Dithiothreitol, a potent synthetic redox potential-delivering compound, when added to growing cells, prevented this induction of NF-kappaB and, simultaneously, chromosomal instability was reduced. Fanconi anemia cells possess low endogenous levels of the naturally occurring antioxidant thioredoxin. Transfection of Fanconi anemia cells with thioredoxin cDNA containing a nuclear localization signal prevented both spontaneous as well as mitomycin C-induced chromosomal instability. A promotor construct with two NF-kappaB binding sites in front of the CAT gene induced little CAT expression in cells with low thioredoxin content in spite of induced NF-kappaB. In cells with higher thioredoxin content CAT expression was increased. Cotransfection of the NF-kappaB-dependent CAT plasmid with the Trx/nuc-plasmid into FA fibroblasts increased the CAT expression to almost that of control cells, indicating that in this model system with diminished thioredoxin content NF-kappaB requires thioredoxin for binding to its specific promotor. Since Fanconi anemia cells have low thioredoxin contents, NF-kappaB-dependent genes are expressed insufficiently. This explains part of the pathophysiological processes observed in Fanconi anemia.     

The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway

Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2.     

Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells

A cDNA library was screened for plant proliferating-cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A lambda gt11 cDNA library was constructed using poly(A)-rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ-1, which contains PCNA-related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29,765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate-starved cells, auxin-starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.     

A DNA double strand break repair defect in Fanconi anemia fibroblasts

Fanconi anemia (FA) is a heterogeneous autosomal recessive disease characterized by congenital abnormalities, pancytopenia, and an increased incidence of cancer. Cells cultured from FA patients display elevated spontaneous chromosomal breaks and deletions and are hypersensitive to bifunctional cross-linking agents. Thus, it has been hypothesized that FA is a DNA repair disorder. We analyzed plasmid end-joining in intact diploid fibroblast cells derived from FA patients. FA fibroblasts from complementation groups A, C, D2, and G rejoined linearized plasmids with a significantly decreased efficiency compared with non-FA fibroblasts. Retrovirus-mediated expression of the respective FA cDNAs in FA cells restored their end-joining efficiency to wild type levels. Human FA fibroblasts and fibroblasts from FA rodent models were also significantly more sensitive to restriction enzyme-induced chromosomal DNA double strand breaks than were their retrovirally corrected counterparts. Taken together, these data show that FA fibroblasts have a deficiency in both extra-chromosomal and chromosomal DNA double strand break repair, a defect that could provide an attractive explanation for some of the pathologies associated with FA.     

Loss of FANCC function is associated with failure to inhibit late firing replication origins after DNA cross-linking

Fanconi anemia (FA) cells are abnormally sensitive to DNA cross-linking agents with increased levels of apoptosis and chromosomal instability. Defects in eight FA complementation groups inhibit monoubiquitination of FANCD2, and subsequent recruitment of FANCD2 to DNA damage and S-phase-associated nuclear foci. The specific functional defect in repair or response to DNA damage in FA cells remains unknown. Damage-resistant DNA synthesis is present 2.5-5 h after cross-linker treatment of FANCC, FANCA and FANCD2-deficient cells. Analysis of the size distribution of labeled DNA replication strands revealed that diepoxybutane treatment suppressed labeling of early but not late-firing replicons in FANCC-deficient cells. In contrast, normal responses to ionizing radiation were observed in FANCC-deficient cells. Absence of this late S-phase response in FANCC-deficient cells leads to activation of secondary checkpoint responses.     

Repair response of human fibroblasts to bleomycin damage

The ability of human fibroblasts to repair the specific types of DNA damage caused by bleomycin (BLM) was examined in whole-cell experiments. The method utilized for analysis was alkaline sucrose-gradient centrifugation of DNA. The results of these studies show that a repair pathway exists for the damage produced in DNA by bleomycin. DNA from BLM-treated cells shows a decrease in molecular weight, caused by chemical or enzymatic incision at sites of drug action. If the drug is removed, the DNA rapidly returns to high molecular weight, demonstrating reformation of damaged DNA. This repair response to BLM-damage was also confirmed in fibroblasts isolated from patients with putative DNA-repair defects. We observed that the response (to BLM) of cells from patients with Fanconi anemia was altered in that the fall in molecular weight of DNA from treated cells was not as great as that observed in other cell strains after drug treatment.     

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.     

Conversion of replicative intermediates in human DNA-repair defective cells

We have examined the conversion of intermediates of DNA replication in normal human skin fibroblasts and fibroblasts isolated from patients with genetic diseases caused by putative DNA repair defects. Experiments were performed in non-transformed, unchallenged cells using alkaline sucrose sedimentation analysis to demonstrate precursor low molecular weight (LMW) DNA molecules which converted into high molecular weight (HMW) DNA with time. Analyses of conversion of replicative intermediates were conducted in cells from patients with ataxia telangiectasia (AT), Fanconi anemia (FA), Bloom syndrome (BS), Cockayne syndrome (CS) and xeroderma pigmentosum (XP). Our studies show that conversion of replicative intermediates occurs in all cell strains examined. However, XP cells (complementation groups A and E) show evidence of abnormalities in the conversion of LMW replicative intermediates, with the most dramatic alterations shown by cells from complementation group A.     

Phosphorylation of fanconi anemia (FA) complementation group G protein, FANCG, at serine 7 is important for function of the FA pathway

Fanconi anemia (FA) is an autosomal recessive disease of cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA cross-linking agents. The molecular mechanism for the disease is unknown as few of the FA proteins have functional motifs. Several post-translational modifications of the proteins have been described. We and others have reported that the FANCG protein (Fanconi complementation group G) is phosphorylated. We show that in an in vitro kinase reaction FANCG is radioactively labeled. Mass spectrometry analysis detected a peptide containing phosphorylation of serine 7. Using PCR-mediated site-directed mutagenesis we mutated serine 7 to alanine. Only wild-type FANCG cDNA fully corrected FA-G mutant cells. We also tested the effect of human wild-type FANCG in Chinese hamster ovary cells in which the FANCG homologue is mutant. Human FANCG complemented these cells, whereas human FANCG(S7A) did not. Unexpectedly, FANCG(S7A) bound to and stabilized the endogenous forms of the FANCA and FANCC proteins in the FA-G cells. FANCG(S7A) aberrantly localized to globules in chromatin and did not abrogate the internuclear bridges seen in the FA-G mutant cells. Phosphorylation of serine 7 in FANCG is functionally important in the FA pathway.     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.     

Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts

A DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [³⁵S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S-phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0.25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.     

Ubiquitin is a heat shock protein in chicken embryo fibroblasts.

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.