National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, USA.

197

Cells from patients with Cockayne syndrome (CS), which are sensitive to killing by UV although overall damage removal appears normal, are specifically defective in repair of UV damage in actively transcribed genes. Because several CS strains display cross-sensitivity to killing by ionizing radiation, we examined whether ionizing radiation-induced damage in active genes is preferentially repaired by normal cells and whether the radiosensitivity of CS cells can be explained by a defect in this process. We found that ionizing radiation-induced damage was repaired more rapidly in the transcriptionally active metallothionein IIA (MTIIA) gene than in the inactive MTIIB gene or in the genome overall in normal cells as a result of faster repair on the transcribed strand of MTIIA. Cells of the radiosensitive CS strain CS1AN are completely defective in this strand-selective repair of ionizing radiation-induced damage, although their overall repair rate appears normal. CS3BE cells, which are intermediate in radiosensitivity, do exhibit more rapid repair of the transcribed strand but at a reduced rate compared to normal cells. Xeroderma pigmentosum complementation group A cells, which are hypersensitive to UV light because of a defect in the nucleotide excision repair pathway but do not show increased sensitivity to ionizing radiation, preferentially repair ionizing radiation-induced damage on the transcribed strand of MTIIA. Thus, the ability to rapidly repair ionizing radiation-induced damage in actively transcribing genes correlates with cell survival. Our results extend the generality of preferential repair in active genes to include damage other than bulky lesions.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome.

Leadon

Cooper²

1993

197

“…A few XP-cell lines have been tested for defects in early steps of excision repair: two were shown to be unable to excise pyrimidine dimers from DNA (4), and another one was reported to be defective in the first step of repair, the endonucleolytic cleavage presumably near UV lesions of the DNA (5). Somatic cell hybridization studies have recently shown that mutations which lead to the XP phenotype fall into five or six complementation groups (6,7). This number exceeds the number of steps postulated for excision repair, suggesting that the repair pathway is regulated in a very complex manner or that there exist multiple, or branched pathways of repair.…”

mentioning

confidence: 91%

An altered apurinic DNA endonuclease activity in group A and group D xeroderma pigmentosum fibroblasts.

Kühnlein¹,

Penhoet²,

Linn³

1976

201

Endonuclease activity upon depurinated DNA was measured in extracts of cultured fibrobrasis from xeroderma pigmentosum patients. Cell lines from complementation groups A, B, C, E, and the XP-variant -had slightly reduced levels of activity, but cell lines from complementation group D had one-sixth of the normal activity. An Cell lines developed from skin biopsies of these individuals have been reported to be defective in excision repair of DNA damage introduced by ultraviolet light or by various chemical agents (2,3). This defect is inferred from a reduction in the overall rate of DNA repair synthesis, and it remains unclear at which step the repair pathway is affected. A few XP-cell lines have been tested for defects in early steps of excision repair: two were shown to be unable to excise pyrimidine dimers from DNA (4), and another one was reported to be defective in the first step of repair, the endonucleolytic cleavage presumably near UV lesions of the DNA (5). Somatic cell hybridization studies have recently shown that mutations which lead to the XP phenotype fall into five or six complementation groups (6, 7). This number exceeds the number of steps postulated for excision repair, suggesting that the repair pathway is regulated in a very complex manner or that there exist multiple, or branched pathways of repair.Several enzyme activities believed to be involved in excision repair have been measured in extracts of XP-cell lines. Pedrini et al. (8) found no significant differences in the levels of polynucleotide ligase activity, DNA polymerase, single-strand exonuclease and double-strand endonuclease. Bacchetti et al. (9) compared endonucleolytic activity specific for UV-irradiated DNA in normal and two XP-cell lines and detected no difference. Under their assay conditions the major UV-specific endonuclease activity in fibroblast extracts did not cleave the DNA near pyrimidine dimers, but at other undetermined lesions. In an attempt to Abbreviation: XP, xeroderma pigmentosum. identify excision repair endonucleases we have also found that extracts from a normal cell line and from the XP-cell lines CRL 1157 and CRL 1160 (see Materials and Methods) contained equal levels of an endonuclease activity which preferentially cleaves UV-irradiated DNA at sites which are about 100 times less frequent than the number of pyrimidine dimers in the DNA. However, these studies led us also to compare endonuclease activity with depurinated DNA as a substrate in extracts from normal and XP fibroblasts. Such an activity has been previously reported in many microorganisms, plants and mammals (10-13). MATERIALS AND METHODSGrowth of Cells and Preparation of Extracts. Fibroblast cell lines from skin biopsies were obtained from the American Type Culture collection (ATCC). Strain numbers, complementation groups and relevant properties are summarized irk Table 1. Cells were grown at 370 in 32 ounce prescription bottles with 50 ml of Dulbecco's modified Eagle's medium (Gibco), supplemented with 15 mM N-2-hydroxyethylpiperazine...

“…To determine the role of unexcised damage in PA synthesis, we compared enzyme induction as a function of dose in XP cells of complementation groups A, C, and D. The strains used have been shown to perform <5%, 16%, and 25%, respectively, ofnormal repair synthesis (29)(30)(31). We found that, the more severe the repair deficiency of XP, the higher the levels of PA induced (Fig.…”

Section: Methodsmentioning

confidence: 99%

Induction of plasminogen activator by UV light in normal and xeroderma pigmentosum fibroblasts.

Miskin¹,

Ben-Ishai²

1981

Normal and DNA repair-deficient human fibroblasts have been used to study induction of plasminogen activator (PA) by DNA damage. UV light induced the synthesis ofPA in skin fibroblasts of all types ofxeroderma pigmentosum (XP) in XP heterozygotes and in human amniotic cells. Enzyme induction was, however, not observed in fibroblasts of normal adults. In classical XP, which are deficient in excision repair, PA synthesis occurred in a narrow range of low-UV fluences. In such strains, the level of enzyme produced was correlated with the extent of repair deficiency. UVfluences required for PA induction in XP variants and XP heterozygotes were at least 10 times those inducing enzyme synthesis in excision-deficient XP. Maximum enzyme induction occurred 48 hr after irradiation, and the highest levels of enzyme produced were 15-20 times those of PA baseline levels. Electrophoretic analysis showed that UV irradiation enhances the synthesis ofthe Mr 60,000 human urokinase-type PA, which is present in low amounts in untreated cells. Our results suggest that PA induction in human cells is caused by unrepaired DNA damage and represents a eukaryotic SOS-like function. In addition, PA induction may provide a sensitive assay for detection of cellular DNA repair deficiencies and identification of XP heterozygotes. DNA damage induces in bacteria the coordinate expression of a set of diverse responses collectively called SOS functions. These functions include the appearance ofrepair and mutagenic activities, inhibition of septation, induction of certain prophages, and production of large amounts of the recA protein (for review, see ref. 1). Induction of SOS functions appears to involve the protease activity of the recA protein, which cleaves cellular repressors ofSOS functions including its own repressor, the lexA gene product (2-5).Several observations suggest that SOS-like activities are also (PA). PA is a highly specific serine protease closely associated with cellular transformation, neoplasia, and tumor promotion (for reviews, see refs. 16, 17). In view of the central role attributed to proteolysis in the regulation of SOS functions in bacteria and because of the possible relation of both PA and SOS-like functions with neoplastic transformation, we further explored the induction of PA in human cells by UV irradiation.In the present work, we studied the role of DNA repair in PA induction by skin fibroblasts from xeroderma pigmentosum (XP) patients. XP belongs to a class of repair-deficient human autosomal recessive disorders that have in common predisposition to cancer and chromosome instability (18,19). XP is clinically characterized by abnormally enhanced sensitivity to sunlight and the appearance of carcinomas in exposed areas of the skin. Two classes ofXP patients can be biochemically identified: those whose cells show defective excision repair of UV light-induced damage to DNA (excision-deficient XP) and patients whose cells are impaired in postreplication repair but exhibit normal excision repair (XP variants) ...