Deoxyribonucleic acid structure in human diploid fibroblasts stimulated to proliferate.

Abstract

When contact-inhibited human diploid fibroblasts that have been maintained on medium containing 0.5% serum are stimulated by 10% serum they leave the G0 state and enter the cell cycle at G1. The onset of DNA synthesis occurs at 12 to 13 h. DNA was isolated from cultures that had been stimulated for 0, 2, 4, 6, 12, 16, 20, 24, and 30 h. These DNA preparations were characterized by sensitivity to a single strand specific nuclease, CsSO4/AgClO4 buoyant density, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1-nuclease. The DNA from 4-h stimulated cultures has an increased amount of single strandedness as indicated by sensitivity to S1-nuclease and buoyant density. The amount of single strandedness increases as the cells enter S-phase and decreases as they enter the G2 period. The DNA isolated from G0 cells behaves as native, duplex DNA with virtually no breaks. The DNA from 2-h and 4-h stimulated cultures contained a significant number of breaks. The number of breaks increases to a maximum in S-phase and declines as the cells enter the G2 period. There are no nuclease-sensitive sites (gaps) in G0 or 2-h cultures. The number of sites increases 4 h after stimulation, reaches a maximum during S-phase, and decreases as the cells enter G2. These results lead us to suggest a chromosomal cycle at the level of DNA itself, whereby structural changes are occurring throughout the entire G1 period, aimed toward the initiation of DNA synthesis. These changes reach a maximum degree during replication and decline as replication is terminated.