Repair of Potentially Lethal Damage by Introduction of T4 DNA Ligase in Eucaryotic Cells

Summary

The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, we find that the PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. Here we show that the concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on the ligase enzyme dose and on ATP concentration in the hypertonic medium. We conclude that T4 DNA ligase is able to repair some of the potentially lethal damage produced by restriction endonucleases in eucaryotic cells.