A system of DNA replication in HeLa nuclei treated with inhibitors of protein synthesis.


An in vitro DNA synthesizing system consisting os isolated nuclei from ...
An in vitro DNA synthesizing system consisting os isolated nuclei from HeLa cells which had been treated with inhibitors of protein synthesis was investigated. Treatment with both 30 microgram/ml cycloheximide and 10 microgram/ml puromycin of S-phase cells reduced the rate of DNA synthesis immediately; however, the overall DNA synthesis continued for up to 4 h with a diminished rate and then ceased. In the nuclei which were isolated from the cells which had been incubated with these drugs for 6 h, little incorporation of [3H]TTP into acid-insoluble materials was observed. Addition of cytosol prepared from cells actively synthesizing DNA induced the incorporation of [3H]TTP in these nuclei, while little induction was observed by the addition of cytosol prepared from drug-treated cells in spite of the fact that the latter cytosol stimulated DNA synthesis in isolated nuclei from non-treated cells. The induced DNA synthesis was shown to require Mg2+, all four deoxyribonucleoside triphosphates and ATP, and to proceed discontinuously. The activity inducing DNA synthesis in drug-treated nuclei fluctuated with the phases in a cell cycle and it was not ascribed solely to DNA polymerase alpha nor to DNA ligase.




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