Unscheduled DNA Synthesis in Cell Lysate System Reflecting DNA Repair In Vivo

Abstract

A cell lysate system by which DNA repair (unscheduled) synthesis induced by DNA damaging agents can be measured at high sensitivity as previously reported (9, 18) was characterized. Time-course experiments in which the in vivo incubation time with hydroxyurea and arabinofuranosyl cytosine after UV irradiation was changed suggested that the number of single strand gaps increased in the presence of these drugs. Alkaline sucrose density gradient analysis of prelabeled DNA revealed that the repair apparatus in the drug-treated cells was not irreversibly impaired. Product DNA in this lysate system was compared with that of an in vitro replication system on alkaline CsCl equilibrium density gradient, and the results showed that DNA synthesis in the lysate system was ‘'repair-type” synthesis. When the cells were labeled with 5-bromo-2′-deoxyuridine (BrdUrd) in vivo and then labeled with [3H] dTTP in vitro, the photolysis of BrdUrd-labeled DNA decreased the molecular weight, indicating that DNA synthesis in the cell lysate was a continuation of DNA repair in vivo. © 1984, Japan Society for Cell Biology. All rights reserved.