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

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.