Comparison of DNA Polymerases α, δ and ε of Mouse Cell Line FM3A and Its Temperature-Sensitive Mutant tsFT20

Ike hat a, H. Comparison of DNA Polymerases α, δ and ε of Mouse Cell Line FM3A and Its Temperature-Sensitive Mutant tsFT20. Tohoku J. Exp. Med., 1994, 172 (1), 65-81 DNA polymerases (pol) α, δ and ε of a mouse cell line FM3A and its temperature-sensitive derivative tsFT20, which is defective in DNA replication at a non-permissive temperature, were purified by chromatographic procedures monitored by a set of relatively specific assays for the respective DNA polymerase activities. The pol ε activity was separated into two fractions with similar enzymatic properties except for their optimal KC1 concentrations and processivities. The fractions of pol δ and ε were not homogeneous, but their identities were confirmed by their sensitivities to DNA polymerase inhibitors, their associated 3' → 5' exonuclease activities, optimal concentrations of salts, dependencies on the proliferating cell nuclear antigen and processivities in polymerization, which also excluded significant contamination with other DNA polymerases. Of the DNA polymerases prepared from tsFT20 cells, only pol α showed greatly decreased activity and remarkable sensitivity to the non-permissive temperature, demonstrating that pol δ and ε, the other polymerases supposed to be involved in nuclear DNA replication, are unequivocally different entities from pol α. The level of pol e activity tsFT20 was also significantly lower than in the parental cells, suggesting cooperation and/or interaction between pol α and ε, and some relevanc of pol ε to DNA replication. DNA polymerase; DNA replication; temperature-sensitive mutant; mammalian cells; protein purification.