DNA polymerase-alpha. Common polypeptide core structure of three enzyme forms from human KB cells.


Aqueous extracts of exponentially growing human KB cells contain three ...
Aqueous extracts of exponentially growing human KB cells contain three quantitatively significant forms of DNA polymerase-alpha activity. The predominant form (about 70% of total cellular activity), which is recovered from the cytoplasm, has been previously purified to near-homogeneity and structurally characterized (Fisher, P. A., and Korn, D. (1977) J. Biol. Chem. 252, 6528-6535). The other two polymerase forms include a minor cytoplasmic activity (about 20% of total cellular activity), DNA polymerase-alpha'; and a species that is tightly associated with detergent-purified nuclei (about 10% of total cellular activity), nuclear DNA polymerase-alpha. We have now purified both minor enzyme species to near-homogeneity and show that they are physically and enzymologically similar to the predominant cytoplasmic species. All three purified forms of KB polymerase-alpha behave as physically homogeneous 7 S species with minimum protomer molecular weights of about 140,000. By examining in vitro 14C-labeled enzyme fractions by high resolution sodium dodecyl sulfate-polyacrylamide gel fluorography, we demonstrate that the three purified species display a similar subunit structure composed of a quartet of polypeptides with molecular masses of 70,000, 65,000, 59,000, and 55,000 daltons, respectively. Extended fluorographic exposures of the gels fail to reveal the presence of any polypeptides larger than 70,000 daltons. It is our present interpretation, based on these and our previous observations, that the four polypeptides reflect microheterogeneity involving two primary species, and we do not yet know how they may associate to form the catalytically active protomer of 140,000 molecular weight. From arithmetical considerations, it is plausible that one or more pairwise combinations of them might suffice to generate the 7 S polymerase activity. These results thus corroborate the principal conclusions of our previous study of the structure of the KB cell cytoplasmic polymerase. In addition, they provide the first comparative analysis of the structure of multiple, operationally distinguishable polymerase-alpha forms obtained from a single tissue, as well as the first description of the purification and structural characterization of a nuclear DNA polymerase-alpha species from any source.




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