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  • Review Article
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Replication fork reversal in eukaryotes: from dead end to dynamic response

Key Points

  • Replication fork reversal is a complex transaction that requires the coordinated unwinding and annealing of parental and newly synthesized strands at the replication fork.

  • Replication fork reversal is a frequent transaction in mammalian cells following different types of genotoxic stress. However, it is usually disfavoured in yeast cells, where alternative events such as re-priming are particularly efficient, and key regulatory factors such as poly(ADP-ribose) polymerase (PARP) are absent.

  • Fork reversal can be seen as a 'double-edged sword'. It contributes to genome stability by promoting DNA damage tolerance and repair, and by protecting chromosome integrity during replication; however, it can also lead to microsatellite instability and unscheduled chromosomal breakage.

  • Various factors can drive fork reversal and fork restoration in vitro, but their activity heavily depends on the molecular features of the DNA substrate and on the presence of auxiliary factors.

  • Fork reversal in vivo is dependent on the homologous recombination factor RAD51, and is regulated by PARP and ATP-dependent RNA helicase Q1 (RECQ1)-mediated fork restart, but additional — mostly unknown — factors are likely to have a role in this process.

  • Fork reversal dynamics have intriguing implications for our understanding of basic replication mechanisms and the DNA damage response. The cellular factors modulating fork remodelling are potentially attractive targets for cancer therapy.

Abstract

The remodelling of replication forks into four-way junctions following replication perturbation, known as fork reversal, was hypothesized to promote DNA damage tolerance and repair during replication. Albeit conceptually attractive, for a long time fork reversal in vivo was found only in prokaryotes and specific yeast mutants, calling its evolutionary conservation and physiological relevance into question. Based on the recent visualization of replication forks in metazoans, fork reversal has emerged as a global, reversible and regulated process, with intriguing implications for replication completion, chromosome integrity and the DNA damage response. The study of the putative in vivo roles of recently identified eukaryotic factors in fork remodelling promises to shed new light on mechanisms of genome maintenance and to provide novel attractive targets for cancer therapy.

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Figure 1: Transactions at replication forks following genotoxic stress.
Figure 2: Physiological roles and pathological consequences of replication fork reversal.
Figure 3: Forming and processing reversed forks in vivo.
Figure 4: Physiological and therapeutic implications of fork reversal.

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Acknowledgements

The authors apologize to all those researchers whose relevant work could not be cited owing to space limitations. The authors would like to thank M. Foiani for useful discussions, D. Branzei and K. Cimprich for critical reading of the manuscript, K. Mutreja for technical assistance, and all current and former members of the Lopes laboratory for useful discussions. K.J.N. is a Marie Skłodowska-Curie Fellow. Work in the Lopes laboratory is supported by the Swiss National Science Foundation, Oncosuisse, the European Research Council and Promedica-UBS Foundation.

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Correspondence to Massimo Lopes.

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Supplementary information

Supplementary information S1 (figure)

Identification of reversed forks in vivo by psoralen crosslinking coupled to transmission electron microscopy. (PDF 1347 kb)

Supplementary information S2 (figure)

Detection of replication fork reversal by 2D gels (PDF 944 kb)

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Glossary

Post-replication repair

A series of repair mechanisms that allow the completion of genome replication following genotoxic stress and include the different DNA damage tolerance strategies.

Topological constraints

Impediments to DNA unwinding owing to the accumulation of DNA supercoiling, which is particularly intense in proximity to active transcription and replication forks.

Template discontinuity

A break in the backbone of the DNA molecule that is subjected to replication, such as single-stranded DNA (ssDNA) nicks or gaps.

Replisome

The large multiprotein complex present at each replication fork that provides all of the biochemical and regulatory activities required for processive and coordinated DNA synthesis.

Nascent strand extrusion

The separation of a newly synthesized strand from its template strand, which may prime annealing with the complementary newly synthesized strand.

Template switching

A DNA damage tolerance mechanism during replication. It involves using the newly synthesized strand as an alternative template for DNA synthesis, instead of the damaged parental strand.

Poly(ADP-ribose)

A polymer of ADP and ribose units synthesized by specialized poly(ADP-ribose) polymerases (PARPs) and deposited on specific protein residues, often serving as a regulatory post-translational modification.

Strand exchange

The process by which a DNA strand anneals with the complementary strand by displacing a previously annealed partner strand.

Holliday junction

A junction between four strands of DNA that serves as a central intermediate in most recombination mechanisms.

DNA translocase

A protein using the energy released from ATP hydrolysis to move along double-stranded or single-stranded DNA.

ssDNA bubbles

Biochemical substrates containing single-stranded DNA (ssDNA) that are used to assess in vitro the pairing of ssDNAs by specific annealing enzymes and activities.

Fanconi anaemia core complex

A large multiprotein complex normally activated by replication perturbation induced by DNA damage; this complex is responsible for the ubiquitylation of additional factors involved in the DNA damage response. Mutations in the factors comprising this complex cause Fanconi anaemia, which is characterized mainly by haematological problems and cancer predisposition.

Fork fusion

The final step of replication termination, when two forks converge from opposite directions, merge and complete DNA synthesis of the intervening sequence.

Primary lesions

DNA lesions directly induced by genotoxic agents, which can potentially be converted into secondary lesions (such as double-strand breaks) by replication or repair mechanisms.

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Neelsen, K., Lopes, M. Replication fork reversal in eukaryotes: from dead end to dynamic response. Nat Rev Mol Cell Biol 16, 207–220 (2015). https://doi.org/10.1038/nrm3935

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