Immunoaffinity-purified DNA polymerase alpha from a mouse temperature-sensitive mutant, tsFT20 strain, is heat-labile.
We have purified DNA polymerase alpha from a temperature-sensitive mutant cell line of mouse FM3A cells, tsFT20, that shows temperature-sensitive activity of DNA polymerase alpha (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1761-1765). The purified enzyme was composed of two polypeptides with the same apparent molecular weights as those of purified DNA polymerase alpha from the parental strain, FM3A (Mr 180,000 and 68,000). Heat inactivation experiments revealed that this purified DNA polymerase alpha from tsFT20 cells was more heat-labile than the wild-type enzyme. We have also purified primase from both ts-FT20 cells and wild-type cells. Both primase fractions consist of two polypeptides with molecular weights of 54,000 and 46,000. No difference was observed between the heat labilities of the primases from tsFT20 cells and wild-type cells. Comparisons of wild-type and mutant polymerase indicated that the temperature-sensitive mutation in DNA polymerase alpha from tsFT20 cells affect the dCTP-binding site of the enzyme. The mutation also changed the optimum pH and the optimum KCl concentration of the enzyme.
Takada-Takayama
R
R
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hanaoka
F
F
Yamada
M
M
Ui
M
M
D000818:Animals; D002846:Chromatography, Affinity; D004257:DNA Polymerase II / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II