Isolation and characterization of replication forks from discrete regions of the polyoma genome

Synthesis of polyoma DNA in nuclei isolated from virus-infected 3T6 mouse fibroblasts leads to the selective labeling of replicative intermediates. Digestion of these replicative intermediates with the restriction endonuclease HpaII resulted in three highly labeled heterogeneous species in addition to the expected full-length fragments. These three species migrated more slowly in agarose than did any of the full-length restriction fragments and were shown to represent families of replication forks by criteria of sensitivity to S1 nuclease, kinetics of labeling both in vitro and in vivo, electron microscopy, and migration behavior during agarose gel electrophoresis. Subsequent digestion with other restriction enzymes showed that the two largest of the three fork bands originated from HpaII fragments 1 and 2. These fragments flank the putative terminus located 180 degrees relative to the origin. The third fork-containing band was less labeled and was derived from fragment 3, which is juxtaposed to the replication origin on the side corresponding to late transcription. A two-dimensional gel system revealed the presence of a fourth fork band, derived from fragment 4, that was obscured by full-length fragments 1 and 2 in the single-dimension electrophoresis. Resolution of the fork families revealed multiple discrete species within the major bands, implying the existence of stops or hesitations during replication of a given region of the genome. This conclusion is consistent with the presence of multiple species upon electrophoresis of the fork bands under denaturing conditions.