TY - JOUR
T1 - 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
AU - Jones, Christopher J.
AU - Cieaver, James E.
AU - Wood, Richard D.
N1 - Funding Information:
The authors thank Maureen Biggerstaff for the preparation of plasmid DNA, Colin Arlett for the 1BR.3N cell line, David Kelsell for fingerprinting, Peter Robins for calf thymus XP-A protein, the ICRF Cell Production Unit for cell culture and D. Szymkowski for comments on the manuscript. This work was supported by the Imperial Cancer Research Fund. J.E.C. is supported by U.S. Department of Energy Office of Health and Environmental Research, contract No. DE-ACO3-76-SF01O12.
PY - 1992/3/11
Y1 - 1992/3/11
N2 - 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 flbroblast 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.
AB - 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 flbroblast 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.
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U2 - 10.1093/nar/20.5.991
DO - 10.1093/nar/20.5.991
M3 - Article
C2 - 1549511
AN - SCOPUS:0026521584
SN - 0305-1048
VL - 20
SP - 991
EP - 995
JO - Nucleic acids research
JF - Nucleic acids research
IS - 5
ER -