Initiation of replication in chromosomal DNA induced by extracts from proliferating cells.

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Abstract
Addition of an extract prepared from a proliferating cell line [murine leukemia P388 cells, mouse fibroblast 3T3 cells, mouse fibroblast SV3T3 cells, chicken embryo fibroblasts transformed by Rous sarcoma virus ts 68 (ts NY68SRA), EL4.BU cells] to nuclei isolated from resting tissues such as frog liver and spleen stimulated DNA synthesis as assayed by [3H]dTTP incorporation. This stimulated incorporation of [3H]dTTP required ATP and depended on Mg2+ and deoxynucleoside triphosphates. Pulse-chase experiments showed that DNA synthesis in this system was discontinuous, resulting in the appearance of approximately 4S fragments and their ligation to yield higher MW DNA. EM analysis of the DNA molecules from the reaction mixture showed that the frequency of replication eyes in the extract-stimulated reaction was 10-fold higher than that observed in controls. All of these results strongly suggest that the extract stimulated initiation of DNA replication in the chromatin of normally resting cells. Preliminary characterization by dialysis, heating and enzyme treatments indicated that the activity is associated with 1 or more proteins of high MW (> 50,000). Comparison of the levels of stimulatory activity in extracts from various mammalian and avian sources showed that the activity was present in cells proliferating either in vivo or in tissue culture. In contrast, extracts from normally resting tissues and cells had no activity. The level of activity present did not appear to be directly related to the levels of DNA polymerase. These results suggest a use for this system in study regulation of the initiation of DNA synthesis and control of the various phases of the cell cycle.