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.     

Deficient DNA end joining activity in extracts from fanconi anemia fibroblasts

Fanconi anemia (FA) is a genetic disorder associated with genomic instability and cancer predisposition. Cultured cells from FA patients display a high level of spontaneous chromosome breaks and an increased frequency of intragenic deletions, suggesting that FA cells may have deficiencies in properly processing DNA double strand breaks. In this study, an in vitro plasmid DNA end joining assay was used to characterize the end joining capabilities of nuclear extracts from diploid FA fibroblasts from complementation groups A, C, and D. The Fanconi anemia extracts had 3-9-fold less DNA end joining activity and rejoined substrates with significantly less fidelity than normal extracts. Wild-type end joining activity could be reconstituted by mixing FA-D extracts with FA-A or FA-C extracts, while mixing FA-A and FA-C extracts had no effect on end joining activity. Protein expression levels of the DNA-dependent protein kinase (DNA-PK)/Ku-dependent nonhomologous DNA end-joining proteins Xrcc4, DNA ligase IV, Ku70, and Ku86 in FA and normal extracts were indistinguishable, as were DNA-dependent protein kinase and DNA end binding activities. The end joining activity as measured by the assay was not sensitive to the DNA-PK inhibitor wortmannin or dependent on the nonhomologous DNA end-joining factor Xrcc4. However, when DNA/protein ratios were lowered, the end joining activity became wortmannin-sensitive and no difference in end joining activity was observed between normal and FA extracts. Taken together, these results suggest that the FA fibroblast extracts have a deficiency in a DNA end joining process that is distinct from the DNA-PK/Ku-dependent nonhomologous DNA end joining pathway.     

A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

Fanconi anemia (FA) is a fatal genetic disorder associated with pancytopenia and cancer. Cells lacking functional FA genes are hypersensitive to bifunctional alkylating agents, and are deficient in DNA double-strand break repair. Multiple genes with FA-causing mutations have been cloned, however, the molecular basis for FA remains obscure. The results presented herein indicate that a Rad50-dependent end-joining process is non-functional in diploid fibroblasts from FA patients. Introduction of anti-Rad50 antibody into normal fibroblasts sensitized them to DNA damaging agents, whereas this treatment had no effect on fibroblasts from FA patients. The DNA end-joining process deficient in FA cells also requires the Mre11, Nbs1 and DNA ligase IV proteins. These data reveal the existence of a previously uncharacterized Rad50-dependent DNA double-strand break repair pathway in mammalian somatic cells, and suggest that failure to activate this pathway is responsible, at least in part, for the defective DNA end-joining observed in FA cells.     

Characterization of a simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.     

Mutation analysis of the Fanconi anemia gene FACC.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disorder characterized by a unique hypersensitivity of cells to DNA cross-linking agents; a gene for complementation group C (FACC) has recently been cloned. We have amplified FACC exons with their flanking intron sequences from genomic DNA from 174 racially and ethnically diverse families in the International Fanconi Anemia Registry and have screened for mutations by using SSCP analysis. We identified eight different variants in 32 families; three were detected in exon 1, one in exon 4, one in intron 4, two in exon 6, and one in exon 14. Two of the eight variants, in seven families, did not segregate with the disease allele in multiplex families, suggesting that these variants represented benign polymorphisms. Disease-associated mutations in FACC were detected in a total of 25 (14.4%) of 174 families screened. The most frequent mutations were IVS4 + 4 A-->T (intron 4; 12 families) and 322delG (exon 1; 9 families). Other, less common mutations include Q13X in exon 1, R185X and D195V in exon 6, and L554P in exon 14. The polymorphisms were S26F in exon 1 and G139E in exon 4. All patients in our study with 322delG, Q13X, R185X, and D195V are of northern or eastern European or southern Italian ancestry, and 18 of 19 have a mild form of the disease, while the 2 patients with L554P, both from the same family, have a severe phenotype. All 19 patients with IVS4 + 4 A-->T have Jewish ancestry and have a severe phenotype.     

Identification of the sporulation gene spoOA product of Bacillus subtilis

A 2.4-kilobase fragment of the Bacillus subtilis chromosome containing the wild-type spoOA gene derived from the phi 105dspoOA+-Bc-1 transducing phage was cloned onto plasmid pBR322 in Escherichia coli. A recombinant plasmid harboring the mutant spoOA12 allele on the 2.4-kilobase insert was also constructed from the phi 105dspoOA12-1 phage DNA and pBR322. Protein products synthesized in response to plasmid DNA in a DNA-directed cell-free system derived from E. coli were analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A protein of approximately 27,500 daltons synthesized with the recombinant plasmid DNA harboring the wild-type spoOA gene as template was not formed with the recombinant plasmid DNA harboring the spoOA12 allele. Since the spoOA12 mutation is a nonsense mutation, we conclude that the 27.5-kilodalton protein is the product of the spoOA gene.     

Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.

The DNA segments containing the ADR1 gene and a mutant allele, ADR1-5c, have been isolated by complementation of function in Saccharomyces cerevisiae. The ADR1 gene is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when S. cerevisiae cells are grown on a nonfermentable carbon source, whereas the ADR1-5c allele allows ADHII synthesis even during glucose repression. A plasmid pool consisting of yeast DNA fragments isolated from a strain carrying the ADR1-5c allele was used to transform a strain containing the adr1-1 allele, which prevents ADHII depression. Transformants were isolated which expressed ADHII during glucose repression. A plasmid isolated from one of these transformants was shown to carry the ADR1-5c allele by its ability to integrate at the chromosomal adr1-1 locus. The wild-type ADR1 gene was isolated by colony hybridization, using the cloned ADR1-5c gene as a probe. The ADR1-5c and ADR1 DNA segments were indistinguishable by restriction site mapping. A partial ADR1 phenotype could be conferred by a 1.9-kilobase region, but DNA outside of this region appeared to be necessary for normal activation of ADHII by the ADR1 gene.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.     

An efficient method to optimize Kluyveromyces lactis gene targeting

Kluyveromyces lactis strains impaired in the nonhomologous end-joining pathway are relevant tools for the homologous integration of exogenous DNA into the genome, as in the mutant strains, close to 100% of the integrants are targeted to the homologous locus, compared with a few per cent for the wild-type recipient. Using a loxP-kanMX-loxP cassette together with a Cre-recombinase plasmid, a nej1∷loxP mutant strain suitable for multiple gene disruption has been constructed. Furthermore, using this strain, PCR-generated constructs with only 50 bp of homologous flanking sequences resulted in efficient exogenous DNA targeting.     

Sequence variations in the Fanconi anaemia gene,FAC: pathogenicity of 1806insA and R548X and recognition of D195V as a polymorphic variant

Fanconi anaemia (FA) is a rare autosomal recessive disorder associated with diverse clinical symptoms, increased chromosomal instability and a marked hypersensitivity to crosslinking agents. At least five complementation groups have been defined, the gene for group C (FAC) being the only FA gene cloned thus far. Several sequence variations have been detected in FA patients, whose assignment to group C, however, had not been ascertained by complementation studies. Using a functional assay, in which we tested the capacity of a variant sequence to correct the defect in FA-C lymphoblasts, we provide evidence for the pathogenic status of 1806insA and R548X and for non-pathogenicity of D195V. Received: 23 April 1996     

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.     

Non-homologous end-joining defect in fanconi anemia hematopoietic cells exposed to ionizing radiation

Fanconi anemia is a genetically heterogeneous recessive disease characterized mainly by bone marrow failure and cancer predisposition. Although it is accepted that Fanconi cells are highly sensitive to DNA crosslinking agents, their response to ionizing radiation is still unclear. Using pulsed-field gel electrophoresis, we have observed that radiation generates a similar number of DNA double-strand breaks in normal and Fanconi cells from three (FA-A, FA-C and FA-F) of the 11 complementation groups identified. Nonsynchronized as well as nonproliferating Fanconi anemia cells showed an evident defect in rejoining the double-strand breaks generated by ionizing radiation, indicating defective non-homologous end-joining repair. At the cellular level, no difference in the radiosensitivity of normal and FA-A lymphoblast cells was noted, and a modest increase in the radiosensitivity of Fanca-/- hematopoietic progenitor cells was observed compared to Fanca+/+ cells. Finally, when animals were exposed to a fractionated total-body irradiation of 5 Gy, a similar hematopoietic syndrome was observed in wild-type and Fanca-/- mice. Taken together, our observations suggest that Fanconi cells, in particular those having nonfunctional Fanconi proteins upstream of FANCD2, have a defect in the non-homologous end-joining repair of double-strand breaks produced by ionizing radiation, and that compensatory mechanisms of DNA repair and/or stem cell regeneration should limit the impact of this defect in irradiated organisms.     

An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function.

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.     

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

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

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

Lack of sensitivity of primary Fanconi's anemia fibroblasts to UV and ionizing radiation

Clinical observations and theoretical considerations suggest some degree of radiosensitivity in Fanconi's anemia (FA), but experimental evidence remains controversial. We tested the sensitivity of primary skin fibroblast cultures from all known FA complementation groups to ionizing radiation and ultraviolet light using conventional cell growth and colony formation assays. In contrast to previous studies, and because FA fibroblasts grow and clone poorly at ambient oxygen, we performed our sensitivity tests under hypoxic cell culture conditions. Fibroblast strains from healthy donors served as negative controls and those from patients with ataxia telangiectasia (AT) and Cockayne syndrome (CS) as positive controls. We observed interstrain variation but no systematic difference in the response of FA and non-FA control fibroblasts to ionizing radiation. After exposure to UV radiation, only complementation group A, G and D2 strains displayed values for colony formation EC50 that were intermediate between those for the negative and positive controls. Because of considerable interstrain variation, minor alterations of the response of individual FA strains to ionizing and UV radiation should be interpreted with caution and should not be taken as evidence for genotype-specific sensitivities of primary FA fibroblasts. All together, our data indicate neither systematic nor major sensitivities of primary FA fibroblast cultures of any complementation group grown under hypoxic cell culture conditions to ionizing or UV radiation.     

Low Levels of RNase H Activity in Escherichia coli FB2 rnh Result from a Single-Base Change in the Structural Gene of RNase H

The DNA coding for RNase H from a mutant strain of Escherichia coli (FB2) was cloned into plasmid pBR322. DNA sequence analysis and the exchange of a portion of the mutant and wild-type genes revealed that a single-base alteration (C-->T) in the coding region of the structural gene for RNase H is responsible for the difference in RNase H activity of the wild-type and mutant cells.     

A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.     

Endonuclease A degrades chromosomal and plasmid DNA of Escherichia coli present in most preparations of single stranded DNA from phagemids.

With E. coli, large and variable amounts of chromosomal and plasmid DNAs are observed in the supernatants of overnight cultures when the cells carry an endA mutation, but are not detected by gel electrophoresis when the cells carry the wild type allele of endA. Significant amounts of nuclease activity in DH11S endA+ supernatants were detected by two simple assays; the rapid degradation of added pBR322 plasmid DNA, as judged by agarose gel electrophoresis, and a decrease of more than 100000 fold in transformation efficiency of the added pBR322 plasmid DNA. By employing isogenic endA mutant and wild type strains of DH11S and DH10B/F' proAB+ laclq Z delta M15, it was shown that detectable levels of chromosomal and plasmid DNAs are observed only in the endA mutant strains. These results indicate that Endonuclease I activity is responsible for degradation of chromosomal and plasmid DNA usually present in preparations of ssDNA. Therefore, a wild type endA gene is useful for the rapid and simple production of highly purified ssDNA from cells containing phagemid vectors.     

Alielic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.