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 alpha, the temperature and pH optima were 38.1 degrees C and 8.0, respectively; for pol beta, these optima were 36.2 degrees C and pH 7.4. Pol gamma showed a pH optimum at 7.7. Optimum activity for both the alpha and beta 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 pol-catalyzed 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 beta-type activity appeared in early S phase, whereas a pol alpha-type activity appeared in late S. During the G1, M, and G2 phases, a background level of pol activity was observed that showed intermediate kinetic properties.
D002453:Cell Cycle; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006367:HeLa Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019384:Nucleic Acid Synthesis Inhibitors; D013696:Temperature
D019384:Nucleic Acid Synthesis Inhibitors; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005033:Ethylmaleimide