Unique DNA repair properties of a xeroderma pigmentosum revertant.

Abstract

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.Xeroderma pigmentosum (XP) is a human recessive disorder that in the homozygote exhibits a large increase in skin cancer induced by sunlight (5). The sensitivity of XP cells in culture to UV light (254 nm) is 5-to 10-fold greater than nQrmal (1,15), and the cells fail to repair a wide range of radiation-and chemically induced DNA damage (5, 10, 27), including (5-5,6-6)cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts, measured by radioimmunoassay (4, 19-21) and enzymatic methods (7,13,14,44). The relative proportions of cyclobutane dimers to other photoproducts determined from their photoreactivation suggest that (6-4) photoproducts may account for as much as 30% of the total lesions (24), and they are highly mutagenic (2). One of the problems encountered in attempts to clone the XP gene results from the reversion of the XP phenotype to UV resistance (16,28,29); therefore, we decided to fully characterize revertants themselves.XP12RO (a simian virus 40-transformed group A cell line), GM637 (a simian virus 40-transformed normal cell line), and normal fibroblasts (HS27 and AG) were grown in Eagle minimal essential medium with fetal calf serum, penicillin, and streptomycin. The XP12RO cell line was cloned by choosing a single colony from among those that grew from a culture of 100 to 200 cells. Large cultures (107 to 108 cells) of XP12RO were exposed to ethyl methanesulfonate at a concentration of 1, 3, or 6 mM in culture medium for 16 h, after which the medium was replaced and the cultures were grown for 1 week. The cultures were subsequently transferred on regular occasions (approximately twice per week), based on the cell density. They were irradiated in phosphate-buffered saline with 1 to 3 J of UV light per m2 (254 nm; 1.3 J/m2 per s) at each transfer. After an accumulated dose of approximately 60 J/m2, the cultures were given a dose of 6.5 J/m2 and allowed to grow for 3 weeks to produce colonies. One colony was chosen for each ethyl methanesulfonate dose and grown into a large culture for subsequent analysis. Cell survival after UV irradiation was determined from the number of macroscopic colonies (>50 cells) formed in 21 days (J. E. Cleaver and G. H. Thomas, J. Invest. Dermatol., in press). Each of the cell lines derived from XP12RO proved to be resistant to UV light (Fig. 1A), and, accordingly, they were defined as revertants (designated XP129, XP322, and * Corresponding author. XP644). When similar-size cultures were not exposed to ethyl methanesulfonate, no surviving colonies were recovered. Revertants showed intermediate sensitivities to 4-nitroquinoline-1-oxide (4NQO) (Fig....

Malondialdehyde, a Product of Lipid Peroxidation, Is Mutagenic in Human Cells

“…tissue source or mechanism of immortalization), the assay was repeated using another set of wild-type and XP-A-deficient fibroblast lines that are both SV40-transformed (26). The same results were obtained, i.e.…”

Mutagenic Lesions in Photocarcinogenesis: The Fate of Pyrimidine Photoproducts in Repair‐Deficient Disorders

“…This mutation appears to reduce the capacity for repair of cyclobutane dimers specifically in inactive regions of chromatin (14,15). We wanted to investigate the intracellular function of XPA by development of an inducible gene expression system with which variations of XPA expression could be related to DNA repair and cell survival.…”

Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.

“…Cells in the classical XP complementation groups and the majority of cellular mutants that are defective in repair of UV-induced DNA damage are defective in repair of both lesions. However, a few rodent and human cell mutants that have altered repair capacities for only one of these photoproducts have been identified (6,13,20,27). These mutants should be useful for evaluating the respective biological roles of the photoproducts and may also provide insight into the mechanism of excision repair in mammalian cells.…”