DNA damage and repair in normal, xeroderma pigmentosum and XP revertant cells analyzed by gel electrophoresis: excision of cyclobutane dimers from the whole genome is not necessary for cell survival.
Human fibroblasts were embedded in microscopic-sized agarose beads and lysed in situ to produce chromosome-sized DNA trapped inside the beads. Conventional alkaline agarose gel electrophoresis of trapped DNA from cells damaged by X-rays before embedding gave mol. wts similar to those obtained with alkaline sucrose gradients. When cells were irradiated with UV light before embedding in agarose and incubated with UV endonuclease to cleave cyclobutane pyrimidine dimer sites, UV damage was detectable by gel electrophoresis over a range of 2.6-52 J/m2 as a linear function of dose, and repair was detectable within 6 h of irradiation. Two independently derived UV-resistant xeroderma pigmentosum (XP) revertants did not remove cyclobutane dimers up to 48 h after irradiation. Incubation of normal and XP revertant cells with cytosine arabinoside after UV irradiation resulted in similar numbers of single-strand breaks; these breaks represent repair of non-dimer photoproducts. Evidently, excision of cyclobutane pyrimidine dimers from the whole genome is not necessary for survival of human cells after UV irradiation; rather, repair of non-dimer photoproducts such as photoproducts in the genome as a whole or cyclobutane dimers in a small number of genes may be more biologically important.
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143-0750.
J E
JE
Cleaver
D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D003561:Cytarabine / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000166:cytology / Q000528:radiation effects; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D014466:Ultraviolet Rays; D014965:X-Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D003561:Cytarabine; D004247:DNA; D006918:Hydroxyurea