Dissociation and reconstitution of the DNA replicase system of HeLa cell nuclei.
The DNA replication system of S-phase HeLa nuclei has been dissociated by cautious extraction at 0 degrees C with 0.25 M NaCl. Replicase activity has been reestablished by recombination of the fractions and reduction of the salt concentration. The reconstituted system, like the starting nuclei, depended on ATP, 4dNTP, MgCl2, the proper ionic strength and the soluble cytoplasmic protein fraction. The activity of the nuclear extract showed a cell cycle dependency and was elevated in the nuclei of cells at the G1 leads to S boundary. In the presence of Mg2+ the major activity of the nuclear extract precipitated during dialysis to reduce the salt concentration; this precipitate exhibited DNA polymerase alpha activity. Chromatography of the active extracts over phosphocellulose separated the replicase supporting factors into three fractions. The major activity eluted in the fraction containing the DNA polymerase alpha activity; the other two active fractions were devoid of polymerase activity. The fraction containing DNA polymerase alpha from the nuclear extracts supported DNA replicase activity in salt-extracted nuclei whereas an equivalent level of DNA polymerase alpha from the cytoplasm was not effective. The data suggest that the DNA polymerase alpha of the salt extracts of S-phase nuclei is either different than the cytoplasmic enzyme or is associated with some essential replicase-supporting factor.
D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D006367:HeLa Cells / Q000201:enzymology*; D046911:Macromolecular Substances; D009994:Osmolar Concentration
D046911:Macromolecular Substances; D004254:DNA Nucleotidyltransferases