In Vitro Deoxyribonucleic-acid-Replicating System Solubilized from Isolated Nuclei of Saccharomyces cerevisiae

Abstract

An in vitro DNA replication system having a high specific activity could be prepared as a clear solution from isolated nuclei of Saccharomyces cerevisiae as described below. The isolated nuclei were treated with 0.4% Nonidet P-40 and precipitated by centrifugation in 20% sucrose. From the precipitate thus obtained, DNA-replicating activity was extracted with 0.5 m KC1. The extract has the ability to incorporate ³H-dTMP into the acid-insoluble fraction (DNA)in the presence of a template DNA, such as pJDB 219 or YRp 7. After 10 min incorporation reaction, in the template DNA, formation of a "replication eye" could be demonstrated under an electron microscope. The sum of the lengths of the eyes was found to be equivalent to the amount of the synthesized DNA calculated from incorporated ³H-dTMP.The specific activity (4.9 nmol dTMP incorporation/mg protein of solubilized fraction/60 min, at 28°C) of the DNA-replicating reaction measured with the present system was approximately 60-fold higher than the highest one previously reported.^{1, 2)}