Intact DNA polymerase alpha/primase from mouse cells. Purification and structure.

Abstract:

A procedure is described for purification of DNA polymerase alpha/primase from cultured mouse lymphoblasts. Approximately 0.5 mg of enzyme, free of detectable contaminants, was obtained from 40 g of cells using seven conventional purification steps. The mouse enzyme contains subunits of 180, 70, 56, and 47 kDa, almost completely intact and similar to the subunit sizes reported for DNA polymerase alpha/primase from Drosophila embryos (Kaguni, L. S., Rossignol, J-M., Conaway, R. C., and Lehman, I. R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2221-2225) and Saccharomyces (Plevani, P., Foiani, M., Valsasnini, P., Badaracco, G., Cheriathundam, E., and Chang, L.M.S. (1985) J. Biol. Chem. 260, 7102-7107). A similar structure has also been inferred for mammalian DNA polymerase alpha/primase; however, previous descriptions of the highly purified mammalian enzyme complex have included substantial evidence of proteolysis and/or partial loss of subunits. In particular, the intact 180-kDa subunit has ordinarily been a minor component, and the molecular mass of the complex and total number of subunits have not been established. Results reported here indicate that the native DNA polymerase alpha/primase consists of one each of the four subunit sizes for a total of 353 kDa, based on estimates from denaturing gels, or 344 kDa when sizes deduced from available nucleic acid sequence data are substituted for three of the four subunits. A figure of 313 kDa was calculated from the sedimentation coefficient (8.9 S) and Stokes radius (81.1 A), the values for which also indicate a frictional ratio of 1.80, corresponding to an axial ratio of approximately 16 and suggesting a highly extended structure.

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