Genetic complementation of UV-induced DNA repair in Chinese hamster ovary cells by the denV gene of phage T4.
The denV gene of phage T4, encoding the pyrimidine dimer-specific DNA repair enzyme endonuclease V, has been introduced by DNA transfection into the UV-sensitive DNA repair-deficient Chinese hamster ovary (CHO) cell line UV5. Transformants were first selected for resistance to the antibiotic G418 conferred by the neo gene from Tn5 carried by the same plasmid. A majority of the isolated G418-resistant UV5 clones also showed an increased resistance to 254-nm UV light. One clone, designated I-A1, was found to have an intermediate level of colony-forming ability after UV irradiation when compared to UV5 and wild-type AA8 cells. A Southern blot showed that I-A1 carries a single integrated intact copy of the denV gene. Alkaline sucrose gradients revealed a dose-dependent appearance of breaks in the DNA of I-A1 cells following UV-irradiation, while unirradiated cells did not exhibit any significant breaks. Analysis of DNA repair by isopycnic sedimentation showed that DNA excision repair by I-A1 was at least equal to the level of repair in AA8 cells. These results show that the prokaryotic denV gene can restore UV repair capabilities in vivo to CHO UV5 cells defective in repair of UV-induced damage.
D000818:Animals; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000096:biosynthesis / Q000528:radiation effects; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics*; D005260:Female; D005814:Genes, Viral; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D010053:Ovary; D013604:T-Phages / Q000235:genetics*; D014162:Transfection; D014466:Ultraviolet Rays
D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)