Supercoiling, knotting and replication fork reversal in partially replicated plasmids

Abstract

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

Direct Evidence for the Formation of Precatenanes during DNA Replication

“…All the immunograms were aligned attending to the mobility of OCs. Note that chloroquine's intercalation has no effect on the electrophoretic mobility of nicked unreplicated molecules (29,37,50).…”

“…This intercalation changes DNA twist. As bacterial plasmids are (Ϫ)-supercoiled, chloroquine removes (Ϫ)-supercoiling first and adds net (ϩ)-supercoiling only after all native (Ϫ)-supercoiling has been removed (37,51). Moreover, it was repeatedly shown that in partially replicated molecules containing three-way forks, overwinding of the unreplicated region transforms three-way into four-way junctions called reversed forks or chicken feet (37, 50 -53).…”

“…For this reason, it would have a profound effect on pBR-terE@StyI but not on pBR-terE@DraI (29,37). We chose 5 g/ml chloroquine as we knew that for pBR322 with a ⌬Lk ϭ 0, this concentration introduces ϳ23 (ϩ)-supercoils.…”

“…Moreover, stalling of the replication fork at the terETus complex only occurs in ϳ80% of the cases. In the remaining ϳ20% (ϳ5 plasmids per cell), the replicating fork goes by terE unimpaired probably due to a failure in the formation of a stable terE-Tus complex (36,37). In these plasmids, replication is completed and segregation follows provided Topo IV is active.…”

Topological locking restrains replication fork reversal

“…The second dimension was in 1% agarose gel in TBE buffer containing increasing concentrations of EthBr run perpendicular with respect to the first dimension. The dissolved agarose was poured around the excised agarose lane from the first dimension, and electrophoresis was at 5 V/cm in a 4°C cold chamber for 10 h. Southern transfer was performed as described (11,21). placed 7 cm above the plastic dish, and the light was filtered through a Pyrex glass to remove radiation Ͻ300 nm.…”