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Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

Abstract

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments1,2. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis3. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

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Figure 1: Observation of replication loops.
Figure 2: Fluorescence imaging of coordinated DNA replication.
Figure 3: Replication loop dynamics depend on primase activity.
Figure 4: Signalling and collision mechanisms both serve to trigger loop release.

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Acknowledgements

We thank J.-B. Lee for technical advice and S. Moskowitz for illustrations. This work was supported by the National Institutes of Health (grants GM-077248 to A.M.v.O. and GM-54397 to C.C.R.) and the National Science Foundation (CAREER grant 0543784 to A.M.v.O.). J.J.L. acknowledges the Jane Coffin Childs Memorial Fund for a postdoctoral fellowship.

Author Contributions S.M.H. performed the single-molecule bead experiments; S.M.H. and J.J.L. performed the single-molecule fluorescence experiments; S.M.H. and M.T. performed the bulk-phase experiments; S.M.H., C.C.R. and A.M.v.O. designed the experiments, analysed the data and wrote the manuscript.

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Correspondence to Antoine M. van Oijen.

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Hamdan, S., Loparo, J., Takahashi, M. et al. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457, 336–339 (2009). https://doi.org/10.1038/nature07512

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