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Research Article

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

Seungwoo Chang, Karel Naiman, Elizabeth S. Thrall, James E. Kath, Slobodan Jergic, View ORCID ProfileNicholas E. Dixon, View ORCID ProfileRobert P. Fuchs, and View ORCID ProfileJoseph J. Loparo
PNAS December 17, 2019 116 (51) 25591-25601; first published December 3, 2019; https://doi.org/10.1073/pnas.1914485116
Seungwoo Chang
aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
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Karel Naiman
bTeam DNA Damage Tolerance, Cancer Research Center of Marseille, CNRS, UMR7258, F-13009 Marseille, France;
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Elizabeth S. Thrall
aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
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James E. Kath
aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
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Slobodan Jergic
cSchool of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia;
dMolecular Horizons, University of Wollongong, Wollongong, NSW 2522, Australia
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Nicholas E. Dixon
cSchool of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia;
dMolecular Horizons, University of Wollongong, Wollongong, NSW 2522, Australia
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  • ORCID record for Nicholas E. Dixon
Robert P. Fuchs
bTeam DNA Damage Tolerance, Cancer Research Center of Marseille, CNRS, UMR7258, F-13009 Marseille, France;
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  • ORCID record for Robert P. Fuchs
  • For correspondence: robert.fuchs@inserm.fr joseph_loparo@hms.harvard.edu
Joseph J. Loparo
aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
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  • ORCID record for Joseph J. Loparo
  • For correspondence: robert.fuchs@inserm.fr joseph_loparo@hms.harvard.edu
  1. Edited by Michael E. O’Donnell, HHMI and The Rockefeller University, New York, NY, and approved November 5, 2019 (received for review August 21, 2019)

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Significance

DNA replication is the high-fidelity process by which cells duplicate their chromosomes prior to cell division. Cellular DNA is constantly damaged, and the resulting DNA lesions can block replication, leading to genome instability and cell death. Cells use multiple pathways to resolve stalled replication. Understanding resolution pathway choice is important because some of these pathways are more likely to introduce mutations than others. In bacterial cells, replication stresses, including antibiotic treatment, lead to mutagenesis, which may contribute to the emergence of antibiotic resistance. In this study, we describe a molecular interaction within the Escherichia coli replication machinery that plays a crucial role in resolution pathway choice, thereby influencing whether lesion-stalled replication is resolved in an error-prone or error-free manner.

Abstract

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

  • DNA replication
  • replication stalling
  • translesion synthesis
  • damage avoidance
  • repriming

Footnotes

  • ↵1S.C. and K.N. contributed equally to this work.

  • ↵2Present address: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, BN1 9RQ Falmer, United Kingdom.

  • ↵3Present address: Marseille Medical Genetics, UMR1251 Aix Marseille Université/Inserm, 13385 Marseille, France.

  • ↵4To whom correspondence may be addressed. Email: robert.fuchs{at}inserm.fr or joseph_loparo{at}hms.harvard.edu.
  • Author contributions: S.C., K.N., E.S.T., R.P.F., and J.J.L. designed research; S.C., K.N., E.S.T., R.P.F., and J.J.L. performed research; S.C., K.N., E.S.T., J.E.K., S.J., N.E.D., R.P.F., and J.J.L. contributed new reagents/analytic tools; S.C., K.N., E.S.T., R.P.F., and J.J.L. analyzed data; and S.C., K.N., E.S.T., J.E.K., S.J., N.E.D., R.P.F., and J.J.L. wrote the paper.

  • The authors declare no competing interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1914485116/-/DCSupplemental.

Published under the PNAS license.

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A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes
Seungwoo Chang, Karel Naiman, Elizabeth S. Thrall, James E. Kath, Slobodan Jergic, Nicholas E. Dixon, Robert P. Fuchs, Joseph J. Loparo
Proceedings of the National Academy of Sciences Dec 2019, 116 (51) 25591-25601; DOI: 10.1073/pnas.1914485116

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A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes
Seungwoo Chang, Karel Naiman, Elizabeth S. Thrall, James E. Kath, Slobodan Jergic, Nicholas E. Dixon, Robert P. Fuchs, Joseph J. Loparo
Proceedings of the National Academy of Sciences Dec 2019, 116 (51) 25591-25601; DOI: 10.1073/pnas.1914485116
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