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, Nicholas E. Dixon, Robert P. Fuchs, and Joseph J. Loparo
PNAS December 17, 2019 116 (51) 25591-25601; first published December 3, 2019 https://doi.org/10.1073/pnas.1914485116

  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.

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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