Multiple forms of nuclear deoxyribonucleic acid polymerases and their relationship with the soluble enzyme.
1. DNA polymerase from nuclear and supernatant fractions of cultured mouse L929 cells was fractionated on columns of Sephadex G-200, Sepharose 4B and of DEAE-cellulose. Several peaks of activity are found on Sephadex chromatography and the distribution of activity between these depends on: (a) the source of the enzyme, i.e. nuclear or supernatant fraction; (b) the mode of extraction of the enzyme from the nucleus; (c) the amount of enzyme applied to the column. 2. The DNA polymerase activity in the lower-molecular-weight peaks (approximate molecular weights are 35000, 70000 and 140000) is firmly bound within the cell nucleus and shows a preference for native DNA as template, whereas the high-molecular-weight peak (peak I, molecular weight 250000 or greater) is found in supernatant fractions and shows greater activity with a denatured DNA template. 3. During periods of DNA synthesis the high-molecular-weight enzyme becomes more firmly bound within the nucleus. 4. Peak I enzymic activity is relatively unstable and is inhibited by thiol-blocking reagents and deoxycholate, but it is stimulated by univalent cations. 5. Very little endonuclease is present in the polymerase preparations, but a very active exonuclease and nucleoside diphosphokinase are present. On Sephadex chromatography, however, it was shown that the immediate precursors for DNA synthesis, at least by peak I enzyme, are the deoxyribonucleoside triphosphates. 6. Attempts to decrease the molecular weight of the peak I enzyme while still retaining activity failed.
D000818:Animals; D002247:Carbon Isotopes; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004247:DNA; D004254:DNA Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003840:Deoxycholic Acid; D004720:Endonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D008566:Membranes / Q000201:enzymology; D051379:Mice; D009691:Nucleic Acid Denaturation; D009711:Nucleotides; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D012995:Solubility; D013439:Sulfhydryl Reagents; D013698:Templates, Genetic; D014316:Tritium
D002247:Carbon Isotopes; D009711:Nucleotides; D013439:Sulfhydryl Reagents; D003840:Deoxycholic Acid; D014316:Tritium; D004247:DNA; D010770:Phosphotransferases; D004254:DNA Nucleotidyltransferases; D004720:Endonucleases; D005092:Exonucleases