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Abstract
Topoisomerase IV (TopoIV) is a vital bacterial enzyme which disentangles newly replicated DNA and enables segregation of daughter chromosomes. In bacteria, DNA replication and segregation are concurrent processes. This means that TopoIV must continually remove inter-DNA linkages during replication. There exists a short time lag of about 10–20 min between replication and segregation in which the daughter chromosomes are intertwined. Exactly where TopoIV binds during the cell cycle has been the subject of much debate. We show here that TopoIV localizes to the origin proximal side of the fork trailing protein SeqA and follows the movement pattern of the replication machinery in the cell.
Highlights
Proper segregation of newly replicated DNA is essential for the viability and genetic stability of all cell types
From kymographs of the fluorescent foci (Fig. 1a) and plots of relative fluorescence intensity according to position along the cell long-axis (Fig. 1b), we found that Topoisomerase IV (TopoIV) had a localization pattern that resembled that of SeqA and the replisome
It is known that DNA segregation occurs gradually during the DNA replication phase[14] and it is known that TopoIV has an important role in unlinking the newly replicated DNA molecules to enable s egregation[6,9]
Summary
Proper segregation of newly replicated DNA is essential for the viability and genetic stability of all cell types. In Escherichia coli two type II topoisomerases are involved in enabling both DNA replication and timely DNA segregation, namely Gyrase and Topoisomerase IV (TopoIV) Both of these enzymes work by first performing a transient double strand break in one molecule, leading a second DNA duplex through the cut and lastly, resealing the cut. They are heterotetrameric structures consisting of GyrA and GyrB subunits or ParC and ParE subunits for Gyrase and TopoIV, respectively. Inhibition of TopoIV using a fluoroquinolone antibiotic, Ciprofloxacin, lead to an increased distance between SeqA and TopoIV, presumably because the TopoIV molecules become “stuck” in DNA ternary complexes, thereby lagging even further behind the replication machinery
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