Comparison of DNA Polymerases .ALPHA., .DELTA., and .EPSILON. of Mouse Cell Line FM3A and Its Temperature-Sensitive Mutant tsFT20.

Abstract

IKEHATA, H. Comparison of DNA Polymerases a, d, and E of Mouse Cell Line FM3A and Its Temperature-Sensitive Mutant tsFT20. Tohoku J. Exp. Med., 1994, 172 (1), [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81] DNA polymerises (pol) a, d and E 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 s activity was separated into two fractions with similar enzymatic properties except for their optimal KC1 concentrations and processivities. The fractions of pol d and 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 tsFT2O cells, only pol a showed greatly decreased activity and remarkable sensitivity to the non-permissive temperature, demonstrating that pol s and E, the other polymerises supposed to be involved in nuclear DNA replication, are unequivocally different entities from poi a. The level of pol E activity tsFT2O was also significantly lower than in the parental cells, suggesting cooperation and/or interaction between pol a and ~, and some relevance of pol E to DNA replication. DNA polymerase; DNA replication; temperature-sensitive mutant; mammalian cells; protein purificationOf the five species of mammalian DNA polymerases (pol) so far known, at least three, pol a, s and E are thought to be involved in nuclear DNA replication (Wang 1991;Araki et al. 1992). The role of pol a in DNA replication is indicated in cytological studies with monoclonal antibodies against poi a (Miller et al. 1985;Kaczmarek et al. 1986), and with a mouse temperature-sensitive cell line tsFT2O (Murakami et al. 1985), which has heat-labile pol a (Takada-Takayama et al. 1991), and in a biochemical study with an in vitro model system for eukaryotic

Quality Control of DNA Polymerase α

“…The tsFT20 cell line, a temperature-sensitive mutant clone identified in a screen of N-methyl-N'-nitro-N-nitroguanidine-treated mouse mammary carcinoma FM3A cells, has been the subject of many studies during the last 30 years ( Fig.6; Eki et al 1986, Eki et al 1987a, Eki et al 1987b, Eki et al 1990, Ikehata 1994, Ikehata et al 1997, Izumi et al 1994, Murakami et al 1985, Pendergrass et al 1994, TakadaTakayama et al 1991, Yamaguchi et al 1995. Compared to parental FM3A cells, this cell line grows normally at 33˚C.…”

Fundamental Aspects of DNA Replication

“…The tsFT20 cell line, a temperature-sensitive mutant clone identified in a screen of N-methyl-N'-nitro-N-nitroguanidine-treated mouse mammary carcinoma FM3A cells, has been the subject of many studies during the last 30 years (Fig. 6; Eki et al 1986, Eki et al 1987a, Eki et al 1987b, Eki et al 1990, Ikehata 1994, Ikehata et al 1997, Izumi et al 1994, Pendergrass et al 1994, Yamaguchi et al 1995. Compared to parental FM3A cells, this cell line grows normally at 33C.…”

Incubation at the Nonpermissive Temperature Induces Deficiencies in UV Resistance and Mutagenesis in Mouse Mutant Cells Expressing a Temperature-Sensitive Ubiquitin-Activating Enzyme (E1)

“…The following ts mutant cell lines were used: ts85 and tsFT5 defective in E1 (9,22,23), tsFT20 defective in Pol ␣ (11,13,24), and tsFT210 defective in p34 cdc2 kinase (21,41,45) at the nonpermissive temperature 39ЊC. All of these mutants were derived from the wild-type clone 28 of the mouse mammary carcinoma suspension cell line FM3A (26).…”