DNA repair synthesis in bleomycin-pretreated permeable HeLa cells

Abstract

To establish an in vitro system for studying DNA repair, bleomycin-induced unscheduled DNA synthesis in permeable HeLa cells was investigated. Permeable HeLa cells were incubated at 0°C for 60 min with 0.11 mM bleomycin, washed to remove free bleomycin and assayed for DNA synthesis. Optimum [ ³ H]deoxythymidine monophosphate incorporation occurred at pH 7.6–8.0 (adjusted at 20°C with Tris-HCI buffer), 3–6 mM MgCl ₂ , 40–60 mM NaCl, and 2.5–5 mM ATP in the presence of four deoxynucleoside triphosphates. The unscheduled nature of DNA synthesis in bleomycin-pretreated permeable cells was confirmed by the BrdUMP density shift technique. Exonuclease III sensitivity of repaired DNA was measured to determine whether or not the completion of repair patches and ligation occurred in bleomycin-pretreated permeable cells. Gap-filling and ligation were suggested to occur in the presence of ATP. Studies using the selective inhibitors (aphidicolin, 2',3'-dideoxythymidine 5'-triphosphate and N-ethylmaleimide) for DNA synthesis showed that DNA polymerases α and β were involved in the repair process. Inhibitor studies suggested that DNA polymerase α plays a preferential role in repair label in the intranucleosomal region of nuclear chromatin and DNA polymerase β in the completion of repair patches in bleomycin-pretreated permeable cells.