One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells.
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 (D0 = 56 J X m-2) than in the other human cell lines tested (normal, ataxia-telangiectasia, Lesch-Nyhan, retinoblastoma)(D0 = 680 J X m-2)(D0 is the dose that reduces the percentage of CAT activity by 63% along the exponential portion of the dose-response curve). The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 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.
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics; D001260:Ataxia Telangiectasia / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D002472:Cell Transformation, Viral; D015500:Chloramphenicol O-Acetyltransferase; D004260:DNA Repair; D004274:DNA, Recombinant / Q000528:radiation effects; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase; D007926:Lesch-Nyhan Syndrome / Q000235:genetics; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000528:radiation effects; D011401:Promoter Regions, Genetic; D011740:Pyrimidine Dimers / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012175:Retinoblastoma / Q000235:genetics; D013539:Simian virus 40; D014158:Transcription, Genetic / Q000528:radiation effects; D014170:Transformation, Genetic; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D010430:Pentosyltransferases; D007041:Hypoxanthine Phosphoribosyltransferase