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© 1996 Oxford University Press

research-article

Stable transformation of xeroderma pigmentosum group A cells with an XPA minigene restores normal DNA repair and mutagenesis of UV-treated plasmids

Scott P. Myrand 1, Robert S. Topping 2 and J.Christopher States 3

Center for Molecular Medicine and Genetics, Wayne State University 2727 Second Avenue, Detroit, MI 48201, USA

3 To whom correspondence should be addressed

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 neor cassette and selected for G418 resistance (2-O-A2), DNA repair-normal human fibroblasts and untransfected XP12BE-SV cells. Colony forming ability after UV irradiation, 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{uparrow}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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