Stable transformation of xeroderma pigmentosum group A cells with an XPA minigene restores normal DNA repair and mutagenesis of UV-treated plasmids.
The ability of an XPA minigene construct to complement the DNA repair defect in xeroderma pigmentosum group A (XP-A) cells was demonstrated. XP-A cells (XP12BE-SV) were stably transformed with an XPA minigene linked to a neomycin resistance (neor) expression cassette. The G418-resistant clone XAN1 was isolated and its DNA repair phenotype compared with XP12BE-SV cells transformed with a cosmid containing a human chromosome 8 gene and a neo(r) cassette and selected for G418 resistance (2-0-A2), DNA repair-normal human fibroblasts and untransfected XP12BE-SV cells. Colony forming ability after UV-irradiated reactivation of a UV-irradiated chloramphenicol acetyltransferase (CAT) expression vector and UV-induced mutagenesis in a supF tRNA shuttle vector (pSP189) were all restored to normal levels in XAN1 cells. In addition, mutation spectra in the supF gene of pSP189 after replication in all four cell lines were compiled at low (100 J/m2) and high (1000 J/m2) UV doses. The majority of mutations were point mutations and these were predominately G:C-->A:T transitions regardless of dose for all cell lines. Dose-dependent differences were observed in the positions of mutation hot spots in pSP189 mutation spectra after replication in all four cell lines. Mutation spectra for XAN1 and GM0637 cells had only minor differences. An increase in the proportion of transversions was observed only in plasmids irradiated with a low UV dose and replicated in XAN1 cells. 2-0-A2 cells were reported to have partial restoration of DNA repair that was later suggested to be caused by a reversion. 2-0-A2 cells were nearly identical to XP12BE-SV cells in all aspects investigated, indicating that transformation to neor had no effect on DNA repair in these cells.
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Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48201, USA.
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D001483:Base Sequence; D002460:Cell Line; D002461:Cell Line, Transformed; D002898:Chromosomes, Human, Pair 8; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000528:radiation effects*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051760:Xeroderma Pigmentosum Group A Protein
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase