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Single-molecule studies reveal the function of a third polymerase in the replisome

Nature Structural & Molecular Biology volume 19, pages 113–116 (2012)Cite this article

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Abstract

The Escherichia coli replisome contains three polymerases, one more than necessary to duplicate the two parental strands. Using single-molecule studies, we reveal two advantages conferred by the third polymerase. First, dipolymerase replisomes are inefficient at synthesizing lagging strands, leaving single-strand gaps, whereas tripolymerase replisomes fill strands almost to completion. Second, tripolymerase replisomes are much more processive than dipolymerase replisomes. These features account for the unexpected three-polymerase-structure of bacterial replisomes.

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Figure 1: TriPol replisomes are more processive than DiPol replisomes.
Figure 2: TriPol replisomes are more efficient on the lagging strand than DiPol replisomes are.
Figure 3: Analysis of ssDNA gaps in lagging strand products.

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Acknowledgements

This work was supported by US National Institutes of Health grant GM38839 to M.O.D. We thank A. van Oijen and D. Fenyo for helpful suggestions. We also thank L. Langston for suggestions and for critical reading of the manuscript.

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Authors and Affiliations

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

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

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  1. Roxana E Georgescu
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  2. Isabel Kurth
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  3. Mike E O'Donnell
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Contributions

R.E.G. and I.K. carried out experiments; R.E.G., I.K. and M.E.O. designed the experiments. R.E.G., I.K. and M.E.O. wrote the manuscript.

Corresponding author

Correspondence to Mike E O'Donnell.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Methods (PDF 314 kb)

Supplementary Video 1

Replication performed by Dipol replisomes. An example of a movie depicting real-time observation of coupled leading/lagging strand replication of a mini-rolling circle substrate by E. coli Dipol replisomes. The force of the hydrodynamic flow pushes the DNA-lipid complex to a diffusion barrier etched in the glass surface and concentrates numerous DNA molecules in the visual field shown here. The width of the visible area in the direction of the flow is 73 μm (equivalent to 220 kb) and the flow direction is from top to the bottom. Individual DNA molecules visualized with the fluorescent dye Yo-Pro1 are stretched by the buffer flow (100 μl/min) and imaged through Total Internal Reflection Fluorescence (TIRF) microscopy. Toward the end of the movie, the buffer-flow is stopped, letting the strands recoil, then the buffer flow is started again. Movie contains circa 7' 30″ of experimental data rendered at 20 frames per second (original data acquisition is 1 frame/s at 100 ms exposure per frame). (MOV 1058 kb)

Supplementary Video 2

Replication performed by Tripol replisomes. The video depicts the recording of a replication reaction performed by Tripol replisomes. The movie contains circa 5' 30″ of experimental data rendered at 20 frames per second (original data acquisition is 1 frame/s at 100 ms exposure for each frame). (MOV 322 kb)

Supplementary Video 3

DNA molecules that harbor duplex regions contain gaps on the same molecule. The video depicts a recording at the end of a replication reaction using a DiPol replisome. The flow of the buffer solution is stopped then restarted, allowing the DNA strands to stretch and then recoil to their point of origin. (MOV 437 kb)

Supplementary Video 4

Use of fluorescent SSB to identify ssDNA in DNA products. The video depicts three successive recordings of different DNA products of DiPol replisomes, in which reactions contained fluorescently labeled SSB. The three successive recordings are easy to identify since they have different dimensions. The videos show that DNA products contain fluorescently labeled E. coli SSB (with Oregon Green488 Maleimide). The duplex DNA is not visualized because Yo-Pro1 is omitted from the buffer flow for these experiments. To distinguish SSB bound to DNA from SSB that binds non-specifically to the surface of the flow cell, the buffer-flow is alternatively stopped and restarted in order to observe the recoiling of the DNA strands. Fluorescent SSB bound to DNA recoils and re-extends in synchrony with the changes in buffer flow (while non-specifically bound SSB does not change position). (MOV 1871 kb)

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Georgescu, R., Kurth, I. & O'Donnell, M. Single-molecule studies reveal the function of a third polymerase in the replisome. Nat Struct Mol Biol 19, 113–116 (2012). https://doi.org/10.1038/nsmb.2179

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