Isolation of the catalytic core of DNA polymerase alpha from rabbit bone marrow

Abstract

Modification of the purification procedures for rabbit bone marrow DNA polymerase [Byrnes, J.J., & Black, V.L. (1978) Biochemistry 17, 4226–4231] has increased the yield and stability of the enzyme thus allowing further purification. In particular, the higher molecular weight form, alpha₁, has been more abundant. Additional purification has been obtained upon phospho-cellulose and chromatofocusing column chromatography. SDS slab gel electro-phoretic analyses of the eluates demonstrate a 135,000 molecular weight polypeptide in nearly pure form which correlates with DNA polymerase activity. Approximately 200,000 nmol of thymidine monophosphate is incorporated into DNA (mg of protein)⁻¹h⁻¹ at 37°C. Similar to DNA polymerase alpha from other sources this enzyme is an acidic protein, is very sensitive to aphid-icolin, and has no detectable 3′ to 5′ nuculease activity.