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

Journal Article


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Abstract


  • 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.

Publication Date


  • 2017

Published In


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.

Scopus Eid


  • 2-s2.0-85019563709

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2113&context=ihmri

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/1087

Has Global Citation Frequency


Start Page


  • e23932-1

End Page


  • e23932-17

Volume


  • 6

Place Of Publication


  • United Kingdom

Abstract


  • 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.

Publication Date


  • 2017

Published In


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.

Scopus Eid


  • 2-s2.0-85019563709

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2113&context=ihmri

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/1087

Has Global Citation Frequency


Start Page


  • e23932-1

End Page


  • e23932-17

Volume


  • 6

Place Of Publication


  • United Kingdom