DNA repair synthesis in HeLa cell lysate.

In order to study the mechanism of DNA repair, we established an in vitro system for repair (unscheduled) DNA synthesis. HeLa cells synchronized at G2-G1 phase were irradiated with ultraviolet light in the presence of two DNA replication inhibitors, hydroxyurea and 1-beta-D-arabinofuranosyl cytosine (araCyt), to reduce the replicative DNA synthesis as much as possible. Hypotonic treatment of the cells was followed by gentle homogenization, and the resulting cell lysate was incubated with [3H]dTTP. The lysate system required all four dXTPs and Mg2+, but required no ATP. The incorporation of [3H]dTTP was dependent on the dose of ultraviolet light, was linear for 2 min, and reached the maximum at 5 min. The presence of hydroxyurea and araCyt during in vivo incubation was necessary for in vitro DNA synthesis. Accumulation of single-strand breaks was observed under these conditions, and this could explain the very high incorporation of [3H]dTTP in this system.