Unscheduled DNA synthesis in cell lysate system reflecting DNA repair in vivo.

Abstract

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.Mechanisms of DNA repair in eukaryotic cells have been studied by various systems and techniques. These can be classified into two categories : in vivo and in vitro studies. The former includes colony formation, measurement of unscheduled DNA synthesis in cells (radioactivity counting and autoradiography), host cell reactivation, and nucleoid sedimentation. In intact cells, elucidation of the mechanisms of DNA repair is frequently hampered by the complexity of the membrane transport of nucleosides, DNA precursor synthesis, and concomitance of DNA replication. Therefore, it is often difficult to obtain clear-cut data. We were able to eliminate these problems in in vitro systems by providing a constant level of DNA precursors and setting conditions more favorable for DNA repair than for replication.