Deoxyribonucleic acid replication in synchronized cultured mammalian cells. I. Time of synthesis of molecules of different average guanine + cytosine content

HeLa and mouse strain L-cells synchronized by release from excess thymidine block were pulse-labeled with [¹⁴C]thymidine or ³²P at intervals throughout the DNA synthetic period. The nuclear DNA was then isolated and fractionated into molecules of different buoyant densities by preparative CsCl density-gradient centrifugation. Early in the DNA synthetic period, DNA species having high buoyant densities had the highest specific activity. These results indicated that, compared to total nuclear DNA, the DNA replicated early in this period has a higher average buoyant density and hence, higher average G + C content. Late in the period, DNA species having low buoyant densities had the highest specific activities. This pattern was maintained when HeLa cells were synchronized by the selective detachment of mitotic cells. It was also observed when HeLa DNA replicated early in the DNA synthetic period was fractionated in alkaline CsCl and when DNA of the lysed nuclei was analyzed without further purification, thus eliminating the possibility of selective losses of DNA during isolation. By labeling with bromodeoxyuridine, the DNA replicated during a selected interval of the DNA synthetic period could be isolated from the total DNA for base composition analysis. HeLa DNA synthesized in this early period had an average G + C content of 43.6% and that synthesized late in the period had an average G + C content of 38.7%. The average G + C content of pulse-labeled randomly growing cells was 40.1%, similar to the base composition of the total DNA. In thymidine-synchronized mouse L-cells, there was a gradual change in the average G + C content of DNA replicated throughout the synthetic period. The replication of DNA of high G + C content in the early part of the period and the replication of DNA of low G + C content in the late part of the period was similar to the pattern observed in HeLa cells. The specific radioactivities of satellite and main band DNA's were measured after 1-hour pulses with ³²P at different times during the DNA synthetic period. The ratio of the specific activity of satellite to main band DNA increased continuously throughout the period. The specific activities of satellite DNA indicated that about 20% of mouse satellite DNA synthesis occurred during the first 2 hours of the period, and about 80% after main band DNA had already reached its maximum rates of synthesis.