Biosynthetic Properties of a Polyoma Nucleoprotein Complex: Evidence for Replication Sites

Abstract

Under normal growth conditions, all of the newly synthesized polyoma deoxyribonucleic acid (py DNA) that could be extracted from infected mouse cell cultures by the Triton procedure of Green, Miller, and Hendler was in the form of a 55 S nucleoprotein complex. Inhibition of protein synthesis by cycloheximide reduced the sedimentation rate of the polyoma complex synthesized during the first hour after addition of the drug to 25 to 35 S . Since the 55 S and the 25 to 35 S complexes each contain closed circular 20 S py DNA, it is suggested that the slower complex contains less protein per DNA molecule and that there is normally a small or unstable pool of protein available for binding to newly replicated py DNA. In the presence of cycloheximide, the newly formed 25 to 35 S complex was not derived from preexisting 55 S complex. Thus, some py DNA which was not solubilized by the Triton method served as a template for replication. Further evidence for the existence of polyoma replication sites is provided by the demonstration that, during the inhibition of protein synthesis, a class of newly replicated py DNA can be solubilized by the sodium dodecyl sulfate procedure of Hirt, but not by the Triton method. It is postulated that continuous protein synthesis is required to release py DNA from replication sites in the form of a Triton-extractable nucleoprotein complex.