A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode

Slobodan Jergic; Nicholas P. Horan; Mohamed Elshenawy; Claire E. Mason; Thitima Urathamakul; Kiyoshi Ozawa; Andrew J. Robinson; Joris M H Goudsmits; Yao Wang; Xuefeng Pan; Jennifer L. Beck; Antoine M. Van Oijen; Thomas L. Huber; Samir Hamdan; Nicholas E. Dixon

doi:10.1038/emboj.2012.347

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode

Slobodan Jergic, Nicholas P. Horan, Mohamed Elshenawy, Claire E. Mason, Thitima Urathamakul, Kiyoshi Ozawa, Andrew J. Robinson, Joris M H Goudsmits, Yao Wang, Xuefeng Pan, Jennifer L. Beck, Antoine M. Van Oijen, Thomas L. Huber, Samir Hamdan, Nicholas E. Dixon

Research output: Contribution to journal › Article › peer-review

71 Scopus citations

Abstract

Processive DNA synthesis by the αÉ"θ core of the Escherichia coli Pol III replicase requires it to be bound to the β 2 clamp via a site in the α polymerase subunit. How the É" proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of É". Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in É" that, in conjunction with the site in α, maintains a closed state of the αÉ"θ-β 2 replicase in the polymerization mode of DNA synthesis. The É"-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states. © 2013 European Molecular Biology Organization.

Original language	English (US)
Pages (from-to)	1322-1333
Number of pages	12
Journal	EMBO Journal
Volume	32
Issue number	9
DOIs	https://doi.org/10.1038/emboj.2012.347
State	Published - Feb 22 2013

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: We thank Michelle Blayney and Linda Jessop for preliminary ESI-MS data. This work was supported by grants from the Australian Research Council, including Fellowships to KO, TH, and NED, and by a KAUST Faculty Initiated Collaborative grant to SMH and NED.

ASJC Scopus subject areas

General Neuroscience
General Biochemistry, Genetics and Molecular Biology
Molecular Biology
General Immunology and Microbiology

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10.1038/emboj.2012.347

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Jergic, S., Horan, N. P., Elshenawy, M., Mason, C. E., Urathamakul, T., Ozawa, K., Robinson, A. J., Goudsmits, J. M. H., Wang, Y., Pan, X., Beck, J. L., Van Oijen, A. M., Huber, T. L., Hamdan, S., & Dixon, N. E. (2013). A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode. EMBO Journal, 32(9), 1322-1333. https://doi.org/10.1038/emboj.2012.347

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title = "A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode",

abstract = "Processive DNA synthesis by the α{\'E}{"}θ core of the Escherichia coli Pol III replicase requires it to be bound to the β 2 clamp via a site in the α polymerase subunit. How the {\'E}{"} proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of {\'E}{"}. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in {\'E}{"} that, in conjunction with the site in α, maintains a closed state of the α{\'E}{"}θ-β 2 replicase in the polymerization mode of DNA synthesis. The {\'E}{"}-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states. {\textcopyright} 2013 European Molecular Biology Organization.",

author = "Slobodan Jergic and Horan, {Nicholas P.} and Mohamed Elshenawy and Mason, {Claire E.} and Thitima Urathamakul and Kiyoshi Ozawa and Robinson, {Andrew J.} and Goudsmits, {Joris M H} and Yao Wang and Xuefeng Pan and Beck, {Jennifer L.} and {Van Oijen}, {Antoine M.} and Huber, {Thomas L.} and Samir Hamdan and Dixon, {Nicholas E.}",

note = "KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: We thank Michelle Blayney and Linda Jessop for preliminary ESI-MS data. This work was supported by grants from the Australian Research Council, including Fellowships to KO, TH, and NED, and by a KAUST Faculty Initiated Collaborative grant to SMH and NED.",

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AU - Jergic, Slobodan

AU - Horan, Nicholas P.

AU - Elshenawy, Mohamed

AU - Mason, Claire E.

AU - Urathamakul, Thitima

AU - Ozawa, Kiyoshi

AU - Robinson, Andrew J.

AU - Goudsmits, Joris M H

AU - Wang, Yao

AU - Pan, Xuefeng

AU - Beck, Jennifer L.

AU - Van Oijen, Antoine M.

AU - Huber, Thomas L.

AU - Hamdan, Samir

AU - Dixon, Nicholas E.

N1 - KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: We thank Michelle Blayney and Linda Jessop for preliminary ESI-MS data. This work was supported by grants from the Australian Research Council, including Fellowships to KO, TH, and NED, and by a KAUST Faculty Initiated Collaborative grant to SMH and NED.

PY - 2013/2/22

Y1 - 2013/2/22

N2 - Processive DNA synthesis by the αÉ"θ core of the Escherichia coli Pol III replicase requires it to be bound to the β 2 clamp via a site in the α polymerase subunit. How the É" proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of É". Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in É" that, in conjunction with the site in α, maintains a closed state of the αÉ"θ-β 2 replicase in the polymerization mode of DNA synthesis. The É"-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states. © 2013 European Molecular Biology Organization.

AB - Processive DNA synthesis by the αÉ"θ core of the Escherichia coli Pol III replicase requires it to be bound to the β 2 clamp via a site in the α polymerase subunit. How the É" proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of É". Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in É" that, in conjunction with the site in α, maintains a closed state of the αÉ"θ-β 2 replicase in the polymerization mode of DNA synthesis. The É"-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states. © 2013 European Molecular Biology Organization.

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JF - EMBO Journal

IS - 9

ER -

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode

Abstract

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