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

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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.

At a glance


  1. The topological problem caused by coupled leading- and lagging-strand polymerases.
    Figure 1: The topological problem caused by coupled leading- and lagging-strand polymerases.

    The figure illustrates the topological problem only for the leading-strand polymerase. a, The two Pols (yellow) are coupled through the clamp loader (green). Pols travel a helical path as they synthesize DNA. As the leading Pol spins, the lagging Pol is dragged around this path, twisting the lagging strand around the leading strand (left). Alternatively, the DNA turns instead (right), generating negative supercoils as indicated by the arrow. In either case, the stress can be released by (b) transient dissociation of the leading Pol or (c) transient dissociation of the lagging Pol, which could re-bind the incomplete Okazaki fragment or a new RNA primer.

  2. Signal release does not require primase and correlates with increasing Okazaki fragment length.
    Figure 2: Signal release does not require primase and correlates with increasing Okazaki fragment length.

    a, The replisome, consisting of DNA helicase (blue), Pol III* (one clamp loader (green) that binds three Pols (yellow); only two Pols are shown for clarity) and β-clamps (red) is assembled on a 5′ biotinylated rolling circle DNA, then attached to beads. After unbound proteins are removed, replication is initiated by adding primase, β, SSB, ATP and α-32P-labelled deoxyribonucleotide triphosphates. Reactions are quenched and treated with S1 to cleave gaps left by signal release, before analysis on alkaline gels. The newly synthesized leading strand (purple) is the template for lagging-strand synthesis (blue), initiated by RNA primers (red). b, Replication reactions using [α-32P]dTTP either before (lane 1) or after (lane 2) S1 analysis, or using [α-32P]dATP (lane 3). c, Reactions in the absence of primase, using 800nM DNA 20-base oligonucleotides to prime the lagging strand. d, Titration of DNA 20-base oligonucleotides into reactions. e, Plot of Okazaki fragment length versus percentage signal release.

  3. Lagging-strand polymerase processivity is restored using a separate DNA molecule.
    Figure 3: Lagging-strand polymerase processivity is restored using a separate DNA molecule.

    a, The replisome is assembled on DNA as in the legend to Fig. 2a. Unbound proteins are washed away before addition of primed φX174 5.4kb ssDNA. b, Reactions are initiated in the presence (lanes 7–12 and 19–24) or absence (lanes 1–6 and 13–18) of primase. Supernatants containing φX174 replication products (lanes 1–12) are separated from bead-bound rolling circle products (lanes 13–24).

  4. Single-molecule total internal reflection fluorescence microscopy.
    Figure 4: Single-molecule total internal reflection fluorescence microscopy.

    a, The replisome is assembled on the rolling circle DNA and attached to a lipid bilayer. Replication is initiated upon flowing replication buffer at 10μlmin−1. b, DNA curtain produced at 10μlmin−1. c, Normalized line plot intensity of a representative DNA strand. Diagrams at the top illustrate dsDNA and ssDNA regions. d, Analysis as in c, but with Pol III* in the buffer flow. e, Quantification of line plot analysis at indicated flow rates (n = 9±s.d. for 10μlmin−1 or n = 13±s.d. for 100μlmin−1). f, DNA curtain at 100μlmin−1. g, Line plot analysis as in c, at 100μlmin−1. h, Histogram relating pixel intensity to dsDNA at 10μlmin−1 (light blue) and 100μlmin−1 (dark blue). i, Scheme at 100μlmin−1.


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  1. The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10065, USA

    • Isabel Kurth,
    • Roxana E. Georgescu &
    • Mike E. O'Donnell


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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