We studied the structure of replication intermediates accumulated by Tus-induced arrest of plasmid DNA replication at termination sites. For intermediates generated both in vitro with purified components and in vivo, superhelical stress is distributed throughout the entire partially replicated molecule; daughter DNA segments are wound around each other, and the unreplicated region is supercoiled. Thus, unlinking of parental DNA strands by topoisomerases can be carried out both behind and in front of the replication fork. We explain why previous studies with prokaryotic and eukaryotic replication intermediates discerned only supercoiling in the unreplicated portion.
Bibliographical noteFunding Information:
We thank Alex Vologodskii for his valuable advice and Tom Hill for promptly sharing plasmids. B. J. P. was supported in part by a NIEHS training grant, and C. J. U. was supported by a Howard Hughes Medical Institute predoctoral fellowship. This work was supported by NIH grants to N. R. C. and K. J. M.