Cell-Cycle Dependence and Properties of the HeLa Cell DNA Polymerase System

Analysis of the properties of the DNA polymerase (pol) system as a function of fundamental factors of the assay environment allowed a rather accurate estimation of its dependence on the HeLa cell cycle. For pol α , the temperature and pH optima were 38.1 degrees C and 8.0, respectively; for pol β , these optima were 36.2 degrees C and pH 7.4. Pol γ showed a pH optimum at 7.7. Optimum activity for both the α and β enzymes was observed at 60 mM Tris. The maximal activity at 36.2 degrees C and pH 7.4 was associated with resistance to N-ethylmaleimide (MalNEt), whereas that at 38.1 degrees C and pH 8.0 was sensitive to MalNEt. Incorporation of [3H]dTTP was maximal after 1 hr of incubation for the former activity and after 4 hr, for the latter. In extracts from cells in early S phase, the pol activity decreased after 1 hr of incubation, was MalNEt-resistant, and was characterized by temperature and pH optima at 36.2 degrees C and 7.4, respectively. In extracts of late S-phase cells, the polcatalyzed incorporation of [3H]dTTP continued after 4 hr of incubation, was MalNEt-sensitive, and was characterized by temperature and pH optima at 38.1 degrees C and 8.0, respectively. Thus, a pol β -type activity appeared in early S phase, whereas a pol α -type activity appeared in late S. During the G1, M, and G2phases, a background level of pol activity was observed that showed intermediate kinetic properties.