Structural relationships between two forms of DNA polymerase ε from calf thymus

G. Siegal, J. J. Turchi, C. B. Jessee, L. M. Mallaber, R. A. Bambara, T. W. Myers

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

We previously reported purification of two forms of DNA polymerase ε from calf thymus (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). We have now used the 'polymerase trap' photolabeling method to identify the polypeptides containing the polymerase active site in each enzyme preparation. The molecular mass of these polypeptides are 210 and 145 kDa for the polymerases now designated ε and ε*, respectively. Renaturation of polymerase activity from denaturing gel electrophoresis corroborates the polymerase trap results. Photolabeling of polymerase fractions suggests that the smaller subunit is derived by proteolysis of the larger subunit during purification. Native sedimentation coefficient measurements of polymerase-containing column fractions further suggest a precursor/product relationship between the two polymerases. Response of polymerization activity to a battery of inhibitors normally used to distinguish mammalian nuclear DNA polymerases was found to be essentially identical for polymerases ε, ε*, and the ε* generated in fractions initially containing ε. These latter results demonstrate that the loss of the protease-sensitive domain of the active site subunit does not affect catalytic function as measured in a standard DNA polymerase assay. The sole apparent functional difference observed here between the ε and ε* forms is evidence that only the full-length ε form can be directly photocrosslinked to dATP, independent of DNA synthesis. Photolabeling of the post-microsomal supernatant fraction from thymus glands obtained from fetal calves reveals the presence of both the ε and ε* polypeptide.

Original languageEnglish (US)
Pages (from-to)3991-3999
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number6
StatePublished - Jan 1 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Siegal, G., Turchi, J. J., Jessee, C. B., Mallaber, L. M., Bambara, R. A., & Myers, T. W. (1992). Structural relationships between two forms of DNA polymerase ε from calf thymus. Journal of Biological Chemistry, 267(6), 3991-3999.