In Vitro Replication of Nuclear Deoxyribonucleic Acid in Isolated Nuclei of Saccharomyces cerevisiae

The incorporation reaction of [3H-methyl]deoxyribothymidine triphosphate (3H-dTTP) into the TCA-insoluble fraction by isolated nuclei of Saccharomyces cerevisiae was characterized as follows: (i) addition of three deoxyribonucleotides (dATP, dGTP and dCTP) is required for maximal incorporation, (ii) the buoyant density of the incorporated products is approximately 1.70, (iii) the incorporation reaction is strongly inhibited by inhibitors of DNA synthesis, (iv) 98% of radio-activities in the products are recovered in the DNA fraction, (v) the products are DNase-sensitive and appear as long fibers at a high frequency, and (vi) the increase in accumulation of small-fragment DNA is time-dependent during the first 3-min incubation, followed by a rapid increase in large-fragment DNAs. Thus, it was concluded that the isolated yeast nuclei have an ability to synthesize or replicate nuclear DNA in vitro. The ability of isolated nuclei was strongly stimulated by cytoplasmic factor(s) in the cellular-soluble fraction prepared from mid-log cells of S. cerevisiae.