Rapid gene-specific repair of cisplatin lesions at the human DUG/DHFR locus comprising the divergent upstream gene and dihydrofolate reductase gene during early G1 phase of the cell cycle assayed by using the exonucleolytic activity of T4 DNA polymerase.
A novel assay to detect strand-specific DNA repair after cellular exposure to cisplatin at IC50 levels, is used to measure rapid repair in the divergent upstream gene (DUG), a human MutS homolog, and in the bidirectional promoter for dihydrofolate reductase gene (DHFR) and the contiguous upstream DUG. Single-stranded DNA capable of hybridizing to gene-specific probes is generated enzymatically by the 3'-5' exonuclease activity of T4 DNA polymerase. The presence of cisplatin lesions inhibit the exonucleolytic activity of T4 DNA polymerase and block the formation of single-stranded DNA. This decreases the amount of complementary sequence produced when assayed by gene-specific probe hybridization. With the progression of repair, increasing quantities of single-stranded DNA become available for probe hybridization. This assay was applied to human A2780 ovarian carcinoma cells treated with cisplatin at the beginning of G1 phase. A dose-response experiment showed that the assay was applicable down to cisplatin concentrations of 2.5 microM. To assay for strand-specific gene repair, the synchronized cells were treated with cisplatin and then allowed time to repair in drug-free medium. Extensive removal of cisplatin lesions after 2 hr of cellular repair during early G1 phase in the DUG and the DUG/DHFR promoter was measured, with no evidence of repair in the unexpressed delta-globin gene. The extent of preferential DNA repair was much more distinct than has been observed previously at high-drug dosage in asynchronous cells.
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224.
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Rampino
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Bohr
D002453:Cell Cycle; D002478:Cells, Cultured; D002945:Cisplatin / Q000633:toxicity*; D004249:DNA Damage; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase; D005914:Globins / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014764:Viral Proteins
D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D005914:Globins; D013762:Tetrahydrofolate Dehydrogenase; D004259:DNA-Directed DNA Polymerase; D002945:Cisplatin