EXPRESSION OF A TRANSFECTED DNA-REPAIR GENE (XPA) IN XERODERMA-PIGMENTOSUM GROUP-A CELLS RESTORES NORMAL DNA-REPAIR AND MUTAGENESIS OF UV-TREATED PLASMIDS

The XPA gene was initially cloned based on the ability of its cDNA to improve survival of cells from xeroderma pigmentosum complementation g roup A (XP-A) patients following irradiation of the cells with UV, We used plasmid host cell reactivation assays to compare UV mutagenesis a nd the proficiency of DNA repair in a cell line from an XP-A patient, XP2OS(SV40), two derivative cell lines stably expressing XPA cDNAs and in a DNA repair proficient human cell line, Expression of XPA protein in XP2OS cells allowed them to repair UV-treated plasmid pRSVCAT, inc reasing activity of the damaged CAT marker gene >100-fold to levels pr oduced by similarly damaged plasmids in normal cells, Expression of th e XPA protein in XP2OS cells improved replication of the UV-treated sh uttle vector pSP189, increasing plasmid survival and decreasing plasmi d mutation frequency to the levels measured in normal cells. The seque nce locations of most mutation hotspots in the plasmid marker gene wer e similar for the three cell lines and the differences did not correla te with the DNA repair status of the cells, This suggests that the loc ation of mutation hotspots is not directly influenced by DNA repair, E xpression of the XPA protein did cause a shift in the types of mutatio ns seen in the plasmid gene, In the XP2OS cells >95% of the plasmid mu tations were G:C-->A:T transition mutations, In contrast, XP2OS cells expressing XPA produced other types of mutations: three times as many transversion mutations and a 12-fold increase in mutations at A:T base pairs, Furthermore, the distribution of these types of mutations was similar to the proportions measured in normal cells, Strikingly simila r patterns of transition and transversion mutations were found by exam ination of reports of XP and non-XP skin carcinomas containing mutatio ns in the p53 tumor supressor gene, suggesting that the repair status of the cells influenced mutagenesis associated with these skin cancers , Our data suggest that loss of XPA gene function may be sufficient to effect the quantitative and qualitative changes in mutagenesis associ ated with the large increase in skin cancers seen in XP-A patients.