DNA Chain Growth in Isolated HeLa Nuclei

A new method for studying DNA chain growth has been used to investigate DNA replication in isolated nuclei. For this purpose, DNA replication was initiated in S-phase cells with bromodeoxyuridine in order to photosensitize active replicons. The nuclei were then isolated and caused to synthesize DNA in vitro with [³H]dTTP as a precursor to label growing chains. Subsequent irradiation with 313-nm light fragments the bromodeoxyuridine-DNA leaders and releases the contiguous, newly replicated DNA chains for analysis by sedimentation through alkaline sucrose gradients. This method of analysis provides data on the length of the DNA chain segments that are actually synthesized in vitro and on the fraction of these DNA chains that extends from bromodeoxyuridine-prelabeled replicons. Segments of 5 to 9 × 10³ nucleotides attached to the bromodeoxyuridine leaders comprised approximately 40 to 50% of the DNA synthesized in the complete subcellular system. The remainder of the DNA consisted of short fragments of 6-13 S that were initiated in vitro, but failed to be ligated to longer strands. The latter appears to accumulate in the subcellular system as the result of a partial interruption of the native DNA replication process. Omission of the 105 000g cytoplasmic supernatant from the reaction mixture reduced the lengths of the DNA segments synthesized in vitro and the amount of DNA that was replicated. No significant amount of repair synthesis occurred on the template DNA and the initiation of replication in new replicons was restricted in the nuclear system. The data, however, support the concept that DNA synthesis in isolated nuclei continues largely from sites that were actively replicating in the living cells and that isolated nuclei provide a system for dissection of the molecular processes involved in the replication of chromosomal DNA.