In-vivo assessment of DNA ligation efficiency and fidelity in cells from patients with Fanconi's anemia and other cancer-prone hereditary disorders

doi:10.1016/0378-4274(93)90064-5

Toxicology Letters

Volume 67, Issues 1–3, April 1993, Pages 309-324

https://doi.org/10.1016/0378-4274(93)90064-5 Get rights and content

Abstract

We developed a host cell DNA ligation assay, in which we transfected linearized plasmid pZ189 into human lymphoblasts or fibroblasts in order to assess the efficiency and accuracy of DNA ligation within these host cells. We used cell lines from patients with Fanconi's anemia and other chromosome breakage or instability syndromes (Bloom's syndrome, ataxia telangiectasia, Werner's syndrome). With the Fanconi's anemia lymphoblast line GM8010 we did not find a reduced, but a slightly hypermutable DNA ligation. Mutation analysis revealed a unique 7.9–12.5-fold increase in insertions or complex mutations. With cells from the other chromosome breakage/instability syndromes we also found a hypermutable and/or reduced DNA ligation. An impaired DNA ligation might be a common molecular mechanism of genetic instability in these disorders.

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Cited by (13)

A DNA double strand break repair defect in Fanconi anemia fibroblasts
2002, Journal of Biological Chemistry
Citation Excerpt :
Regardless of the nature of the defect, the deficiency in DNA double strand break repair observed in FA fibroblasts may provide an attractive explanation for some of the pathologies associated with FA. Although this conclusion is derived from studies performed on fibroblast cells and may not be applicable to all cell types, evidence from lymphoblasts derived from FA patients also indicates a deficiency in DNA double strand break repair (20, 21, 48). Additionally, an examination of both fibroblasts and lymphoblasts derived from FA patients has revealed no distinct differences in sensitivities to DNA-damaging agents or chromosomal instability, the two main cellular features of FA (16, 19, 49, 50).
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.
Genotoxicity and mutagenicity of the α,β-unsaturated carbonyl compound crotonaldehyde (butenal) on a plasmid shuttle vector
1998, Mutation Research - DNA Repair
Crotonaldehyde is an α,β-unsaturated carbonyl compound and an important environmental and industrial toxic substance. Its mutagenic and carcinogenic properties are related to its reactivity to DNA, where it forms different guanine adducts. In order to study the mutagenic consequences of this agent in intact human cells, we treated the shuttle vector plasmid pZ189 with different doses of crotonaldehyde at 37°C for 2 h and then transfected the such damaged plasmid into the normal human lymphoblast cell line GM0621. Within these host cells the guanine adducts are repaired and the plasmids replicated by cellular enzymes. After 2.5 days replicated plasmids were purified from the cells and plasmid survival was quantitated by transformation ability. With increasing doses of crotonaldehyde, we found a significant decline of plasmid survival, reflecting a pronounced genotoxicity of crotonaldehyde-induced DNA damage in intact human cells. Using the plasmid encoded mutagenesis marker gene supF, we were able to screen for mutants and determine mutation frequency in recovered plasmids. A significant increase in mutation frequency with increasing doses of crotonaldehyde reflects mutagenicity of crotonaldehyde-induced DNA damage. Base sequence analysis of recovered mutants revealed 39% point mutations, 46% deletions, and 15% insertions and inversions. Most of the point mutations (82%) were located at G:C base pairs, which is well explained by the DNA damage profile of crotonaldehyde. Among deletions we found a frequent reoccurrence of two hot spot deletions, representing 62% of all deletions. The sites of breakpoints of these deletions hot spots and of other deletions within the plasmid were also found to be sites of DNA breaks, directly induced by crotonaldehyde, as seen in an endlabeled plasmid fragment, treated with crotonaldehyde. Further analysis of the flanking sequences around the deletion breakpoints revealed a high frequency of four different kinds of short sequence homologies of up to eight base pairs.
DNA repair fine structure in Werner's syndrome cell lines
1996, Experimental Cell Research
Werner's syndrome (WS) is a human segmental progerioid disorder with an autosomal recessive pattern of inheritance. Patients with WS exhibit a number of symptoms resembling a premature aging phenotype. We have examined the fine structure of the DNA repair of UV-induced cyclobutane pyrimidine dimers in Epstein–Barr virus (EBV)-transformed WS lymphoblastoid cell lines and in a primary WS fibroblast cell line. The repair was measured at the level of the gene and also in the general genome. Gene-specific and strand-specific DNA repair was measured in the actively transcribed genes dihydrofolate reductase (DHFR), c-myc,and p53, and in the transcriptionally inactive regions, δ globin and the X-linked 754 domain. Both gene-specific repair and strand-specific repair were deficient in the transformed WS lymphoblastoid cell lines compared to normal controls. In normal cells, repair in the transcribed strand was 25 (4 h), 43 (8 h), and 72% (24 h); in the WS cells on average, repair in the transcribed strand was 18 (4 h), 27 (8 h), and 44% (24 h). However, in the primary WS fibroblast cell line, we found a pattern of preferential gene repair which was similar to that in normal human cells. In contrast to cells from patients with the gene-specific repair deficient disease Cockayne's syndrome, which show greatly delayed RNA synthesis recovery after UV irradiation, the WS cells had normal recovery of RNA synthesis. The DNA repair results differ for the different cell types, and our findings thus do not establish a general DNA repair phenotype for WS cells. The fibroblasts had proficient repair, but in the WS lymphoblasts we find a deficiency in DNA repair which could contribute to the reported hypermutability in these cells. The lymphoblasts are, however, transformed cells, and it raises the concern that biological findings in transformed cells may not reflect the situation in primary cells.
Hypermutable ligation of plasmid DNA ends in cells from patients with werner syndrome
1994, Journal of Investigative Dermatology
Werner Syndrome is a rare autosomal recessive disorder characterized by an increased cancer risk and by symptoms suggestive of premature aging. Cells from these patients demonstrate a typical pattern of chromosomal instability and a spontaneous hypermutability with a high rate of unusually large deletions. We have studied the in vivo DNA ligation in three lymphoblast cell lines from Werner syndrome patients and three from normal donors. In our host cell ligation assay we transfected linearized plasmid pZ189 and measured the amount of plasmid DNA ends rejoined by these host cells as the ability of the recovered plasmid to transform bacteria. A mutagenesis marker gene close to the ligation site allowed screening for mutations. Subsequent mutation analysis provided information about the accuracy of the ligation process. The cells from Werner syndrome patients were as effective as normal cells in ligating DNA ends. However, mutation analysis revealed that the three Werner syndrome cell lines introduced 2.4–4.6 times more mutations (p < 0.001) than the normal cell lines during ligation of the DNA ends: the mutation rates were 69.4, 97.2, and 58.7%, as compared to 23.6, 21.7, and 24.4% in the normal cell lines. These increased mutation frequencies in plasmids ligated during passage through Werner syndrome cells were mainly due to a significant (p <0.001) increase in deletions. This error-prone DNA ligation might be responsible for the spontaneous hypermutability and the genomic instability in Werner syndrome cells and related to the apparently accelerated aging and high cancer risk in affected patients.
Fanconi anaemia and leukaemia - Clinical and molecular aspects
2004, British Journal of Haematology
Reduced joining of DNA ends correlates with chromosomal instability in three melanoma cell lines
2003, Tumor Biology

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In-vivo assessment of DNA ligation efficiency and fidelity in cells from patients with Fanconi's anemia and other cancer-prone hereditary disorders

Abstract

Genomics

J. Am. Acad. Dermatol.

Cell

Mutat. Res.

Gene

Mutat. Res.

Mutat. Res.

Mutat. Res.

Cell

Cloning of cDNAs for Fanconi's anemia by functional complementation

Nature

Cloning of a candidate gene for ataxia-telangiectasia group D

Am. J. Hum. Genet.

Fanconi anemia: a pleiotropic mutation with multiple cellular and developmental abnormalities

Ann. Genet.

Deficiency of DNA ligase activity in Fanconi's anemia

Hum. Genet.

Rejoining of DNA double-strand breaks in human fibroblasts and its impairment in one ataxia telangiectasia and two Fanconi strains

J. Supramol. Struct. Cell. Biochem.

Ataxia telangiectasia: an overview

Breast and other cancers in families with ataxia telangiectasia

N. Engl. J. Med.

Cancers in 44 families with ataxia-telangiectasia

J. Natl. Cancer Inst.

Enhanced chromosome breakage in blood lymphocytes after G2 phase X-irradiation, a marker of the ataxia-telangiectasia gene

J. Natl. Cancer Inst.

On the nature of a defect in cells from individuals with ataxia-telangiectasia

Science

Elevated spontaneous mutation rate in Bloom syndrome fibroblasts

The effects of Bloom's syndrome fibroblasts on genetic recombination and mutagenesis of herpes simplex type 1

Somatic Cell Mol. Genet.

Altered DNA ligase I activity in Bloom's syndrome cells

Nature

DNA Ligase I deficiency in Bloom's syndrome

Nature