Cyclic Template Research Articles

Unwinding of replicating polyoma virus DNA

Replicating polyoma virus DNA (DNA II∗, Bourgaux, Bourgaux-Ramoisy & Dulbecco, 1969) labelled with [ 3H]thymidine either in the growing chains, in the template chains, or in both, was prepared and studied by dye-buoyant density gradient centrifugation and velocity sedimentation in neutral and alkaline sucrose solutions. Purified DNA II∗ was also examined under the electron microscope. Molecules having two cyclic template chains similar to those observed for replicating SV40 DNA were identified. Physical characterization of these chains directly demonstrated that they are unwound during replication. Hence, the covalently-closed template duplex exhibits negative supercoils in neutral solution.