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Single-molecule visualization of fast polymerase turnover in the bacterial replisome

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posted on 2024-11-16, 03:45 authored by Jacob LewisJacob Lewis, Lisanne SpenkelinkLisanne Spenkelink, Slobodan Jergic, Elizabeth A Wood, Enrico Monachino, Nicholas Horan, Karl E Duderstadt, Michael M Cox, Andrew Robinson, Nicholas DixonNicholas Dixon, Antonius van OijenAntonius van Oijen
The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.

Funding

Functional Dissection of the Bacterial Replisome

Australian Research Council

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Under the hood: single-molecule studies of multi-protein machines

Australian Research Council

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History

Citation

Lewis, J. S., Spenkelink, L. M., Jergic, S., Wood, E. A., Monachino, E., Horan, N. P., Duderstadt, K. E., Cox, M. M., Robinson, A., Dixon, N. E. & van Oijen, A. M. (2017). Single-molecule visualization of fast polymerase turnover in the bacterial replisome. eLife, 6 e23932-1-e23932-17.

Journal title

eLife

Volume

6

Language

English

RIS ID

114714

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