Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase α-activity

Abstract

tsFT20 cells, which have temperature-sensitive DNA polymerase α-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase α in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [ 3H]thymidine incorporation. At 39 °C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 °C, but could grow at 37 °C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 °C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 °C→39 °C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase α plays a key role in DNA replication in mammalian cells.