The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (؉)-⌬Lk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (؉)-⌬Lk were not supercoiled nor were the replicated arms interwound in precatenanes. The electrophoretic mobility of these molecules indicated that they have no net writhe. Instead, the (؉)-⌬Lk is absorbed by a regression of the replication fork. As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at the replication fork, which is locally identical to a Holliday junction in recombination. We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visualized the structure with scanning force microscopy. We discuss possible physiological implications of the junction for stalled replication in vivo.

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.

DNA gyrase and topoisomerase IV (Topo IV) have distinct roles as unlinking enzymes during DNA replication despite 40% sequence identity between them. DNA gyrase unlinks replicating DNA by introducing negative supercoils while Topo IV decatenates the two daughter molecules. For this study, we measured the rates of unlinking of various topoisomers of DNA by DNA gyrase and Topo IV. Each enzyme has marked preferences for certain strand-passage reactions. DNA gyrase is a relatively poor decatenase, catalyzing strand-passage events that result in supercoiling at rates several orders of magnitude faster than those causing decatenation. Topo IV, in contrast, decatenates linked circles 10 -40 times more quickly than it removes the intramolecular crossings from supercoiled DNA. Supercoiled catenanes are unlinked at an even more increased rate by Topo IV. Thus, the supercoils augment decatenation rather than compete with catenane crossings for their removal. Knot crossings and the crossings of multiply interlinked catenanes are also preferentially removed by Topo IV. This ability of Topo IV to selectively unlink catenated molecules mirrors its key role in decatenation of replicating chromosomes in vivo.

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