Enhanced pyrimidine dimer removal in repair-proficient murine fibroblasts transformed with the denV gene of bacteriophage T4.
The denV gene of bacteriophage T4, which encodes the pyrimidine dimer-specific repair enzyme endonuclease V, was introduced into murine fibroblasts with normal rodent pyrimidine dimer repair capabilities. Endonuclease V recognizes ultraviolet radiation (UVR)-induced pyrimidine dimers and produces single-strand breaks adjacent to the dimers. These nicks may serve as substrates to initiate excision repair of pyrimidine dimers by endogenous enzymes. In the present study, murine fibroblasts stably transfected with denV were able to remove 50-80% of UVR-induced pyrimidine dimers, while control cells removed only about 20% of dimers under the same conditions of pyrimidine dimer induction and repair. For both control and denV-transfected cells, repair continued for at least 24 h after exposure. When removal of UVR-induced photoproducts was initiated by endogenous excision repair mechanisms, an average of 38 nucleotides were replaced per dimer removed, as determined by bromouracil photolysis; denV-initiated excision repair, on the other hand, resulted in removal of an average of 6 nucleotides per dimer repaired. The enhanced pyrimidine dimer repair capabilities conferred by denV gene expression did not appear to improve post-UVR survival.
Kusewitt
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Center for Photomedicine, Lovelace Medical Foundation, Albuquerque, NM 87108.
Ley
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Henderson
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D000818:Animals; D015139:Blotting, Southern; D001976:Bromouracil / Q000378:metabolism; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics* / Q000502:physiology / Q000528:radiation effects; D004277:DNA, Single-Stranded / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D005347:Fibroblasts; D007700:Kinetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013604:T-Phages / Q000235:genetics; D014162:Transfection / Q000235:genetics
D004277:DNA, Single-Stranded; D011740:Pyrimidine Dimers; D001976:Bromouracil; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)