RAPID GENE-SPECIFIC REPAIR OF CISPLATIN LESIONS AT THE HUMAN DUG DHFRLOCUS COMPRISING THE DIVERGENT UPSTREAM GENE AND DIHYDROFOLATE-REDUCTASE GENE DURING EARLY G(1) 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 expo sure to cisplatin at IC50 levels, is used to measure rapid repair in t he divergent upstream gene (DUG), a human MutS homolog, and in the bid irectional promoter for dihydrofolate reductase gene (DHFR) and the co ntiguous upstream DUG. Single-stranded DNA capable of hybridizing to g ene-specific probes is generated enzymatically by the 3'-5' exonucleas e activity of T4 DNA polymerase. The presence of cisplatin lesions inh ibit the exonucleolytic activity of T4 DNA polymerase and block the fo rmation of single-stranded DNA. This decreases the amount of complemen tary sequence produced when assayed by gene-specific probe hybridizati on. With the progression of repair, increasing quantities of single-st randed DNA become available for probe hybridization. This assay was ap plied to human A2780 ovarian carcinoma cells treated with cisplatin at the beginning of G(1) phase. A dose-response experiment showed that t he assay was applicable down to cisplatin concentrations of 2.5 mu M. To assay for strand-specific gene repair, the synchronized cells were treated with cisplatin and then allowed time to repair in drug-free me dium. Extensive removal of cisplatin lesions after 2 hr of cellular re pair during early G(1) phase in the DUG and the DUG/DHFR promoter was measured, with no evidence of repair in the unexpressed delta-globin g ene. The extent of preferential DNA repair was much more distinct than has been observed previously at high-drug dosage in asynchronous cell s.