Effect of DNA polymerase on nuclei from different phases of cell cycle.

Abstract

DNA replication was investigated in isolated HeLa cell nuclei from different phases of the cell cycle. The addition of S‐phase cytoplasm to S‐phase nuclei restored the ability of these nuclei to synthesize DNA. The S‐phase cytoplasm requirement could be replaced by partially purified calf‐thymus DNA polymerases A and B. The activity of both the S‐phase cytoplasm and purified DNA polymerases in the nuclear system was ATP‐dependent. Using nuclei prelabelled with bromodeoxyuridine and CsCl equilibrium centrifugation, the DNA synthesis in vitro was shown to be of the replicative, not repair type when supported by either S‐phase cytoplasm or DNA polymerase A. DNA polymerase B, but not DNA polymerase A, was capable of using G1 nuclei to synthesize DNA; however, this synthesis was not ATP‐dependent. Gel‐filtration chromatography of HeLa cell‐free extracts have shown that DNA polymerases B and C are present in S‐phase and missing in G1‐phase cells.