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 l inked to a neomycin resistance (neo(r)) expression cassette. The G418- resistant clone XAN1 was isolated and its DNA repair phenotype compare d with XP12BE-SV cells transformed with a cosmid containing a human ch romosome 8 gene and a neo(r) cassette and selected for G418 resistance (2-0-A(2)), DNA repair-normal human fibroblasts and untransfected XP1 2BE-SV cells. Colony forming ability after UV irradiation, reactivatio n of a UV-irradiated chloramphenicol acetyltransferase (CAT) expressio n vector and UV-induced mutagenesis in a supF tRNA shuttle vector (pSP 189) were all restored to normal levels in XAN1 cells, In addition, mu tation spectra in the supF gene of pSP189 after replication in all fou r cell lines were compiled at low (100 J/m(2)) and high (1000 J/m(2)) UV doses. The majority of mutations were point mutations and these wer e predominately G:C-->A:T transitions regardless of dose for all cell lines, Dose-dependent differences were observed in the positions of mu tation hot spots in pSP189 mutation spectra after replication in all f our cell lines, Mutation spectra for XAN1 and GM0637 cells had only mi nor differences, An increase in the proportion of transversions was ob served only in plasmids irradiated with a low UV dose and replicated i n XAN1 cells. 2-0-A(2) cells were reported to have partial restoration of DNA repair that was later suggested to be caused by a reversion, 2 -0-A(2) cells were nearly identical to XP12BE-SV cells in all aspects investigated, indicating that transformation to neo(r) had no effect o n DNA repair in these cells.