Repair of damaged DNA by extracts from a xeroderma pigmentosum complementation group A revertant and expression of a protein absent in its parental cell line.
Cells derived from individuals with mutations in the xeroderma pigmentosum complementation group A gene (XP-A gene) are hypersensitive to UV light and have a severe defect in nucleotide excision repair of damaged DNA. UV-resistant revertant cell lines can arise from XP-A cells in culture. Cells of one such revertant, XP129, were previously shown to remove (6-4) photoproducts from irradiated DNA, but to have poor repair of cyclobutane pyrimidine dimers. To analyze the biochemical nature of the reversion, whole cell extracts were prepared from the SV40-immortalized fibroblast cell lines XP12RO (an XP-A cell line), the revertant XP129 (derived from XP12RO), and 1BR.3N (from a normal individual). The ability of extracts to carry out repair synthesis in UV-irradiated DNA was examined, and immunoblots were performed using antiserum that recognizes XP-A protein. XP12RO extracts exhibited a very low level of repair and no detectable XP-A protein, but repair activity could be conferred by adding purified XP-A protein to the reaction mixture. XP129 extracts have essentially normal repair synthesis consistent with the observation that most repair of UV-irradiated DNA by extracts appears to occur at (6-4) photoproducts. An XP-A polypeptide of normal size was present in XP129, but in reduced amounts. The results indicate that in XP129 a mutational event has converted the inactive XP12RO XP-A gene into a form which expresses an active XP-A protein.
Jones
C J
CJ
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
Cleaver
J E
JE
Wood
R D
RD
D000595:Amino Acid Sequence; D002457:Cell Extracts; D002460:Cell Line; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005816:Genetic Complementation Test; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051760:Xeroderma Pigmentosum Group A Protein
D002457:Cell Extracts; D004268:DNA-Binding Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein