Reduction of DNA-polymerase β Activity of CHO Cells by Single and Combined Heat Treatments

Summary

The effect of single and combined heat treatments on the activity of DNA polymerase β was studied in CHO cells. The activity of polymerase β was determined by measuring the amount of [³H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase α. Biphasic response curves were obtained for all temperatures tested (40–46°C) showing the sensitivity to decrease during heating. A constant activation energy of E_a = 120 ± 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43°C with E_a = 360 ± 40 kcal/mole or E_a = 130 ± 20 kcal/mole for temperatures below or above 43°C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermotolerance was induced by a priming heat treatment at 43°C for 45 min followed by a time interval at 37°C for 16 h or by a preincubation at 40°C for 16 h, respectively, the thermal sensitivity of polymerase β was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase β to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase β activity, confirming earlier studies on this subject.