Regulation of DNA synthesis by the higher-order chromatin structure

Mouse erythroleukemia cells were treated with the topoisomerase II poison V P-16, the intrastrand crosslinking agent cis-DDP, and the ribonucleotide re ductase inhibitor hydroxyurea, In all cases, the rate of DNA synthesis decr eased as a result of the treatment, To study the mechanism of inhibition of DNA chain elongation, we determined DNA synthesis in a cell-free replicati on system containing isolated nuclei and cytoplasmic extracts. The rate of DNA synthesis in the reactions containing nuclei isolated from untreated ce lls and extracts from cells treated with the three drugs were slightly redu ced and did not show significant differences between the drugs. In the syst ems containing nuclei from cells treated with cis-DDP, DNA synthesis was ag ain slightly inhibited; synthesis in nuclei treated with hydroxyurea was en hanced, and synthesis in the systems containing nuclei from cells treated w ith 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 brea ks in the DNA, As VP-16 and sonication mediate changes in chromatin topolog y, these results suggest that, along with the trans-acting signal transduct ion pathways, there is a topologic mechanism for regulation of DNA synthesi s in the S phase of the cell cycle.