Identification of a polypeptide component of mouse myeloma DNA polymerase γ

Abstract

Mouse myeloma DNA polymerase γ was extensively purified to a final specific activity of 156 000 units (nmol dTMP incorporation per h) per mg protein on (rA)_n · (dT)_12–18 as a template primer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein fractions obtained by DNA-cellulose column chromatography revealed that the amount of a polypeptide of $\text{M}{}_{\mathrm{r}}\text{= 47 000}$ changed proportionally with DNA polymerase γ activity. A minor polypeptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 140 000}$ also seemed to change with the enzyme activity, but other polypeptides did not. Analysis by ¹²⁵I-labeled peptide mapping indicates that the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide in the mouse myeloma DNA polymerase γ preparation is structurally related to the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide of chick embryo DNA polymerase γ (Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol. Chem. 255, 7002–7009). An antibody against chick embryo DNA polymerase γ cross-reacted with the mouse enzyme, indicating a structural relationship between avian and murine enzymes. Since the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide accounts for 31.4% of total protein in the purified preparation, the specific activity is estimated to be about 490 000 units per mg of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide. The rate of poly(dT) elongation by the purified enzyme was 1 260 nucleotides per min. This value is in the same range as the turnover number (1 530 nucleotides per min per enzyme molecule) which is calculated from the “expected” specific activity with respect to the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide and the molecular weight ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 188000}$ on the assumption of a tetrameric structure of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide). Results indicate that the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide is a component of the mouse myeloma DNA polymerase γ.