Characterization of the replicative intermediates of polyoma virus

Abstract

The replicative intermediate molecules of polyoma DNA form a heterogeneous band in CsClEtBr density gradients with densities ranging from slightly lower than that of the closed circular viral DNA through that of the nicked form of the DNA. In this gradient the molecules are fractionated according to the extent of replication. Material with a buoyant density closest to the closed circular DNA has replicated the least while that in the region of the nicked DNA has replicated the most. The replicative intermediate DNA sediments heterogeneously in neutral sucrose gradients with sedimentation coefficients ranging from 21 to 25 S. In the electron microscope most of the replicative intermediates have one superhelical region and two relaxed regions. In alkaline sucrose gradients the replicative intermediates separate into parental and daughter strands: The daughter strands are linear molecules of less than or equal to one genome length. The parental strands behave as a cyclic coil in which the extent of topological winding between the two strands is less than that of the mature viral DNA. We present a model for the replication of circular polyoma DNA which involves repeated cycles of nicking and sealing of the parental strands. These swivel points may be introduced by the DNA untwisting enzyme.