Comparison of DNA polymerases alpha, delta, and epsilon of mouse cell line FM3A and its temperature-sensitive mutant tsFT20.
DNA polymerases (pol) alpha, delta and epsilon 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 epsilon activity was separated into two fractions with similar enzymatic properties except for their optimal KCl concentrations and processivities. The fractions of pol delta and epsilon 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 alpha showed greatly decreased activity and remarkable sensitivity to the non-permissive temperature, demonstrating that pol delta and epsilon, the other polymerases supposed to be involved in nuclear DNA replication, are unequivocally different entities from pol alpha. The level of pol epsilon activity tsFT20 was also significantly lower than in the parental cells, suggesting cooperation and/or interaction between pol alpha and epsilon, and some relevance of pol epsilon to DNA replication.
Ikehata
H
H
Cellular Physiology Laboratory, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
D000818:Animals; D004257:DNA Polymerase II / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004258:DNA Polymerase III; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D051379:Mice; D008809:Mice, Inbred C3H; D009154:Mutation; D013696:Temperature; D014407:Tumor Cells, Cultured
D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase