Characterization of DNA polymerase α activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase α activity at restrictive temperatures

Abstract

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase α activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase α activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase α from tsFT20 cells cultured at a permissive temperature (33 °C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 °C) for 8 h (phase II). The activity of DNA polymerase α from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase α from tsFT20 cells cultured at 33 °C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 °C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 °C. DNA polymerase α from the revertants, which can grow at 39 °C and exhibit a partial recovery in heat stability of DNA polymerase α activity, showed an intermediate response to glycerol, between those of DNA polymerase α from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 Cells.