Mutation and expression of the XPA gene in revertants and hybrids of a xeroderma pigmentosum cell line.
A series of ultraviolet (UV)-resistant cell lines have been generated from a UV-sensitive XP group A cell line homozygous for a stop codon (TGA) in the chromosome 9 XPA gene. Three lines generated by chemical mutagenesis acquired the ability to excise (6-4) photoproducts but not cyclobutane dimers from the whole genome; two lines generated by a fusion procedure with hamster cells acquired the ability to excise both (6-4) photoproducts and cyclobutane dimers from the whole genome. A central region of the hamster XPA gene was cloned and sequenced. With the use of species-specific primers in the polymerase chain reaction, we found that the hybrid cell lines do not contain a hamster XPA gene. Sequence analysis showed that all of the UV-resistant cell lines contain reversions of the human stop codon, resulting in missense mutations (glycine or leucine for arginine) or wild-type sequences. The concentration of XPA protein in revertant cell lines was about one-half that in normal cells, which would be expected from heterozygous cells; there was no evidence that the mutant proteins were less stable than the wild-type proteins. These results are consistent with the idea that the XPA protein initiates repair by binding to damaged sites with various affinities, depending on the photoproduct and the transcriptional state of the region. A concentration of XPA protein near 50% is needed before repair can proceed into nontranscribed regions of the genome. The revertant cell lines represent a class of missense mutations in the XPA gene that may have altered specificity and that can be used to understand some of the regulatory differences in repair of photoproducts in various regions of the genome.
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143-0750.
J E
JE
Cleaver
M
M
McDowell
C
C
Jones
R
R
Wood
D
D
Karentz
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002461:Cell Line, Transformed; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011740:Pyrimidine Dimers / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein