Structural and enzymological characterization of immunoaffinity-purified DNA polymerase α·DNA primase complex from KB cells

Scott Wong, L. R. Paborsky, P. A. Fisher, T. S. Wang, D. Korn

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Abstract

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase α·DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase α·DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase α activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase α antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase α in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of ≥ 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the 'microheterogeneity' typically exhibited by polymerase α peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase α polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase α epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.

Original languageEnglish (US)
Pages (from-to)7958-7968
Number of pages11
JournalJournal of Biological Chemistry
Volume261
Issue number17
StatePublished - 1986
Externally publishedYes

Fingerprint

DNA Primase
KB Cells
DNA-Directed DNA Polymerase
Peptides
Peptide Mapping
Phosphoproteins
Protein Subunits
Monoclonal Antibodies
Proteins
Threonine
Immunoassay
Serine
Half-Life
Epitopes
Molecular mass
Purification
Antigens
Amino Acids
Enzymes
Degradation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase α·DNA primase complex from KB cells. / Wong, Scott; Paborsky, L. R.; Fisher, P. A.; Wang, T. S.; Korn, D.

In: Journal of Biological Chemistry, Vol. 261, No. 17, 1986, p. 7958-7968.

Research output: Contribution to journalArticle

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