One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells.

Abstract

We have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. We studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines (Do = 56 J m-2) than in the other human cell lines tested (normal, ataxia-telangiectasia, Lesch-Nyhan, retinoblastoma) (Do = 680 J m-2) (Do is the dose that reduces the percentage of CAT activity by 63% along the exponential portion of the dose-response curve). The Do of the CAT inactivation curve was 50 J-m2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the Do data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA. We found that expression of CAT activity was much more sensitive to UV inactivation in xeroderma pigmentosum cells than in repair-proficient cells. In the xeroderma pigmentosum cells the UV-inactivation target size for cat gene expression was independent of plasmid size or promoter. Measurement of T4 endonuclease V-sensitive sites in irradiated plasmids indicates that one pyrimidine dimer inactivates a target close to the size of the putative cat transcriptional unit.MATERIALS AND METHODS Cells. The human SV40-transformed and primary skin fibroblast cell lines studied are indicated in Table 1. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 20 mM L-glutamine and 10o fetal calf serum.The ataxia-telangiectasia and retinoblastoma cells were grown in minimal essential medium (Eagle) with the same supplements. Cells were grown at 37°C in a CO2 concentration sufficient to keep the pH of the growth medium constant at 7.35.Plasmids. pSV2catSVgpt [7268 base pairs (bp)], pSV2cat (5020 bp), and pRSVcat (5027 bp) [a generous gift from B. Howard, constructed as described (4-7)] contain the bacterial cat gene coding for CAT (Fig. 1). The plasmid pSV2catSVgpt also contains the bacterial gpt gene coding for xanthine phosphoribosyltransferase. The ...

The xeroderma pigmentosum group C gene leads to selective repair of cyclobutane pyrimidine dimers rather than 6-4 photoproducts

“…To generate only CPD lesions, the nonreplicating pRSVcat reporter gene plasmid (24,25) was irradiated with 313-nm monochromatic UVB light (2 J͞m 2 per s) in the presence of 10 Ϫ2 M acetophenone (Sigma) for 1 h under anoxic conditions (28,29). To induce only 6-4PP lesions, the plasmid was irradiated with 1,000 J͞m 2 UVC (1.6 J͞m 2 per s) and subsequently treated with photolyase (PharMingen; 1 ml of 0.05 mg͞ml pRSVcat ϩ 1 l of 5 mg͞ml photolyase) and 405-nm monochromatic blue light (4.5 J͞m 2 per s) for 1 h to remove UVC-induced CPD lesions (30,31).…”

“…We constructed a partially corrected (XP4PA-SE1) and a fully corrected (XP4PA-SE2) cell line by stable transfection of an XPC cell line, XP4PA-SV-EB, with the plasmid pXPC3, which contains XPC cDNA. The ability to repair UV-induced DNA damage was assessed by UV cell survival (24), a plasmid host cell reactivation assay (25), and directly with a photoproduct removal ELISA and specific mAbs (26,27). Increased, but still subnormal, XPC protein levels led to a partial functional correction in XP4PA-SE1 by reconstituting cyclobutane pyrimidine dimer (CPD) but not 6-4 photoproduct (6-4PP) repair in the cells' global genome.…”

DNA‐mediated gene transfer as an indicator of DNA damage and its repair by recipient cells

“…The decrease in transformation efficiency observed when ara-C is present 22-24 h post-transfection, implying that such inhibition of DNA polymerase c~ prevents ex-pression, is consistent with relatively late replication of the cellular DNA with incorporated viral TK gene [l,ll]. Evidence for the role of DNA repair in transfection has been previously obtained using repairdeficient xeroderma pigmentosum cells as recipients in transfection assays with transient expression of psVZcat as an end point [12,13]. In both cases, reduced expression of ultravioletdamaged plasmid was observed in xeroderma pigmentosum cells compared to normal recipients.…”

Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

“…Production of UV-resistant colonies was easily quantified for cDNA-cosmid overlap of >300 bp in exon 8. From these lines of evidence, cDNA-cosmid complementation almost certainly occurs by a conventional mechanism of homologous recombination followed by DNA integration (43) (5,11,24,25,35,39,40,49 (7,31). The cloned S. cerevisiae RAD2 gene functionally complements S. pombe mutant radl3 (38).…”

“…The CAT reporter gene has been used extensively to monitor ER in situ for cultured cells (25,41,49,50,68). UV-irradiated aliquots of plasmid pSVCATgpt were introduced into UV135 and 38.4.4 cells and the cDNA-cosmid transformants as described in Materials and Methods.…”