Cleavage of Replicative Intermediates of Simian Virus 40 Deoxyribonucleic Acid by the Restriction Endonuclease of Escherichia coli B

Abstract

Abstract Replicating DNA molecules of SV40 labeled with [3H]thymidine in the newly synthesized strands were examined by sedimentation in neutral and alkaline sucrose gradients after cleavage by the Escherichia coli B restriction endonuclease. This nuclease creates 1 double strand break per superhelical SV40 DNA molecule. An intermediate in the cleavage of labeled replicating molecules was identified with a sedimentation coefficient of 18.5 S at neutral pH. The final product sedimented as a band with a broad peak between 11 S and 14 S in neutral sucrose gradients. Since linear, duplex SV40 DNA sediments at 14 S in neutral sucrose, the sedimentation characteristics of the final cleavage products were inconsistent with those expected for molecules containing between 1 and 2 times the mass of SV40 DNA. When young or partially (l50%) replicated and nearly fully (g80%) replicated molecules were cleaved by the nuclease, the newly synthesized strands in each case remained intact as judged by alkaline sucrose sedimentation. Since cleavage of replicating molecules by this enzyme occurs only in the unreplicated region, the site of cleavage may be near a specific initiation site for DNA replication or at the replication fork. Replicating molecules prepared with [14C]thymidine in the template strands and [3H]thymidine in the newly synthesized strands revealed a dissociation of the newly synthesized strands from the template strands after cleavage. This result suggests that the template strands must be intact at or very near either the initiation site or the replication fork in order to stabilize the secondary structure of the replicating molecule.