Mouse erythroleukemia cells were treated with the topoisomerase II poison VP-16, the intrastrand crosslinking agent cis-DDP, and the ribonucleotide reductase inhibitor hydroxyurea. In all cases, the rate of DNA synthesis decreased as a result of the treatment. To study the mechanism of inhibition of DNA chain elongation, we determined DNA synthesis in a cell-free replication system containing isolated nuclei and cytoplasmic extracts. The rate of DNA synthesis in the reactions containing nuclei isolated from untreated cells and extracts from cells treated with the three drugs were slightly reduced and did not show significant differences between the drugs. In the systems containing nuclei from cells treated with cis-DDP, DNA synthesis was again slightly inhibited; synthesis in nuclei treated with hydroxyurea was enhanced, and synthesis in the systems containing nuclei from cells treated with VP-16 was significantly reduced. DNA synthesis was reduced to the same extent in a system containing nuclei isolated from untreated cells that had been briefly sonicated to introduce a limited number of double-strand breaks in the DNA. As VP-16 and sonication mediate changes in chromatin topology, these results suggest that, along with the trans-acting signal transduction pathways, there is a topologic mechanism for regulation of DNA synthesis in the S phase of the cell cycle.