"Cytoplasmic" deoxyribonucleic acid polymerase. Structure and properties of the highly purified enzyme from human KB cells.
The freshly prepared crude cytoplasmic fraction of aqueously extracted KB cells contains a single major species of DNA polymerase activity (DNA polymerase C) that sediments homogeneously in low ionic strength sucrose gradients with a peak at 10.8 S. The enzyme activity from frozen crude extracts sediments heterogeneously under these conditions with peaks at 8.4 and 10 S. In 0.45 M salt-containing gradients all of the polymerase activity is recovered as a single 6.4 S species. When purified to a specific activity of 7,300, DNA polymerase C sediments in low ionic strength gradients as a single species of 6.5 S. From combined sedimentation and gel filtration analysis, we estimate the molecular weight of the active protomeric species of the polymerase to be about 170,000. Under no conditions of ionic strength does the enzyme disaggregate to active species smaller than 6.4 to 6.5 S. Sodium dodecyl sulfate-polyacrylamide gel analysis of the most highly purified enzyme fractions reveals two major protein bands of 87,000 and 175,000 daltons, respectively. These data suggest that DNA polymerase C contains an 87,000-dalton component and permit the interpretation that the active protomer of Mr equal 170,000 may be a dimer. The purified enzyme shows maximal activity with gapped duplex DNA and has an absolute requirement for 3'-hydroxyl termini. It utilizes initiated polydeoxynucleotide templates poorly and initiated polyribonucleotide templates not at all. Although the polymerase is inhibited by PPi it has only minimal ability to promote PPi exchange (0.8% of the polymerase activity). The purified enzyme is free of endonuclease and exonuclease activities (less than or equal to 0.003% of the polymerase activity) and demonstrates no primer-template-dependent conversion of substrate dNTP to free dNMP during the polymerization reaction. Finally, DNA polymerase C does not excise misparied primer termini from a synthetic homopolymer primer-template but can utilize such termini as initiation sites, although at a very slow rate.
D002460:Cell Line; D003593:Cytoplasm / Q000201:enzymology* / Q000648:ultrastructure; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D013045:Species Specificity; D013942:Thymine Nucleotides / Q000378:metabolism
D013942:Thymine Nucleotides; D008345:Manganese; D011189:Potassium Chloride; D004254:DNA Nucleotidyltransferases; D008274:Magnesium