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A solution to release twisted DNA during chromosome replication by coupled DNA polymerases


Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands1. However, coupled replication presents a largely unrecognized topological problem. Because DNA polymerase must travel a helical path during synthesis, the physical connection between leading- and lagging-strand polymerases causes the daughter strands to entwine, or produces extensive build-up of negative supercoils in the newly synthesized DNA2,3,4. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is unknown. Here we examine the dynamics of the Escherichia coli replisome, using ensemble and single-molecule methods, and show that the replisome may solve the topological problem independent of topoisomerases. We find that the lagging-strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome because of its contact with the leading-strand polymerase. This behaviour, referred to as ‘signal release’, had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments5,6,7. However, we observe that signal release is independent of primase and does not seem to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.

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Figure 1: The topological problem caused by coupled leading- and lagging-strand polymerases.
Figure 2: Signal release does not require primase and correlates with increasing Okazaki fragment length.
Figure 3: Lagging-strand polymerase processivity is restored using a separate DNA molecule.
Figure 4: Single-molecule total internal reflection fluorescence microscopy.


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We thank L. Langston, A. Libchaber and B. Michel for suggestions on the manuscript. We are also grateful to the National Institutes of Health (GM 38839) for supporting this work.

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I.K. and M.O.D. conceived the project, I.K. and R.G. performed experiments, and I.K., R.G. and M.O.D. designed the experiments, analysed data and wrote the paper.

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Correspondence to Mike E. O'Donnell.

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The authors declare no competing financial interests.

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Kurth, I., Georgescu, R. & O'Donnell, M. A solution to release twisted DNA during chromosome replication by coupled DNA polymerases. Nature 496, 119–122 (2013).

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