Transient and stable complementation of ultraviolet repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4.
In this paper we report both transient and stable complementation of pyrimidine dimer repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4, coding for endonuclease V, a dimer-specific DNA glycosylase. Cotransfection with pRSVdenV in SV40-transformed XP12RO(M1) cells (complementation group A) restored transient expression of an indicator plasmid (pRSVcat) bearing a UV-inactivated chloramphenicol acetyltransferase (cat) gene. In addition, XP12RO(M1) clones stably transformed by pRSVdenV-SVgpt expressed transient chloramphenicol acetyltransferase activity when transfected with UV-inactivated pRSVcat plasmid. These clones also showed partial restoration of colony forming ability and excision repair synthesis after UV irradiation. Immunofluorescence, using an endonuclease V polyclonal antibody, showed the presence of the phage glycosylase in stably transformed xeroderma pigmentosum cells. The cotransfection assay affords a rapid, sensitive procedure to screen for functional cloned DNA repair genes and to test mutant cells for the deficiency of specific steps in DNA repair, such as incision.
D002478:Cells, Cultured; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics*; D005814:Genes, Viral; D005816:Genetic Complementation Test; D006801:Humans; D014162:Transfection; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)