Replication of polyoma DNA in isolated nuclei

It is proposed that the nuclei in vitro retained the capacity to elongate replicative intermediate molecules present at the time of their isolation but only had a very limited ability to initiate the replication of new polyoma DNA molecules.

Abstract

Nuclei isolated from polyoma virus-infected mouse fibroblast 3T6 cells incorporated radioactive [3H]dTTP into the replicative intermediate of polyoma DNA. The replicative intermediate was characterized by centrifugation through neutral and alkaline sucrose gradients, isopycnic banding in a propidiumiodide/cesium chloride gradient, by chromatography on benzoylated naphthoylated DEAE-cellulose and DNA-DNA hybridization. A small but significant amount of radioactivity was incorporated into mature polyoma DNA. The incorporation of radioactivity into polyoma DNA required the presence of dATP, dCTP and dGTP and was stimulated by ATP, Mg2+ and monovalent cations. A substantial part of the radioactivity was incorporated into short chains during the first five minutes of incubation while after 30 minutes most of the incorporation had occurred into longer chains which, in an alkaline sucrose gradient, sedimented close to 16 s, the sedimentation coefficient of unit-length linear polyoma DNA. The time-course of the reaction was studied at 25, 30 and 35 °C. The incorporation was non-linear at all temperatures but reached a similar plateau value in each case. It is proposed that the nuclei in vitro retained the capacity to elongate replicative intermediate molecules present at the time of their isolation but only had a very limited ability to initiate the replication of new polyoma DNA molecules.