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The impact of replication stress on replication dynamics and DNA damage in vertebrate cells

Nature Reviews Genetics volume 18, pages 535–550 (2017)Cite this article

Subjects

Key Points

  • The progression of replication forks is impaired when they encounter intrinsically hard-to-replicate sequences (such as repeats and secondary structures) or the transcription machinery, or when DNA precursors become limiting.

  • Upon fork slowing, cells maintain their replication rate by adjusting the density of initiation events to fork speed, and vice versa. This regulatory process is known as compensation.

  • Fork slowing becomes pathological when it reaches the threshold that exhausts the pool of latent origins, resulting in compensation failure.

  • Activation of the ATM pathway by DNA lesions elicits moderate fork slowing and the compensatory activation of latent origins.

  • The activation of the DNA damage checkpoint both facilitates repair and helps replication to proceed across intrinsically hard to replicate or damaged sequences upon firing of extra-origins.

  • Oncogene overexpression promotes reactive oxygen species (ROS) overproduction and upregulation of cyclin-dependent kinase (CDK), resulting in DNA damage, unscheduled activation of structure-dependent nucleases, precursor shortage and subsequent genome instability.

Abstract

The interplay between replication stress and the S phase checkpoint is a key determinant of genome maintenance, and has a major impact on human diseases, notably, tumour initiation and progression. Recent studies have yielded insights into sequence-dependent and sequence-independent sources of endogenous replication stress. These stresses result in nuclease-induced DNA damage, checkpoint activation and genome-wide replication fork slowing. Several hypotheses have been proposed to account for the mechanisms involved in this complex response. Recent results have shown that the slowing of the replication forks most commonly results from DNA precursor starvation. By concomitantly increasing the density of replication initiation, the cell elicits an efficient compensatory strategy to avoid mitotic anomalies and the inheritance of damage over cell generations.

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Figure 1: Consequences of perturbations of fork progression.
Figure 2: Hard-to-replicate sequences.
Figure 3: Impact of CHK1 or WEE1 deficiency on replication dynamics.

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Acknowledgements

M.D.'s team is supported by the French Association pour la Recherche sur le Cancer (grant ARC- S1220130607073), Agence Nationale de la Recherche (grant ANR-13-BSV6-0008-01) and Institut National du Cancer (grant INCa-2013-103). A.N.'s team is supported by the grant ANR-14-CE35-0003-02.

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Authors and Affiliations

  1. IFOM, the FIRC institute of Molecular Oncology, DNA metabolism laboratory, Via Adamello 16, Milan, 20139, Italy

    Hervé Técher

  2. Institut Gustave Roussy, UMR 8200 (CNRS), 114 Rue Edouard Vaillant, Villejuif, 94800, France

    Stéphane Koundrioukoff & Michelle Debatisse

  3. University Pierre & Marie Curie, Sorbonne Universités, 4 Place Jussieu, Paris, 75005, France

    Stéphane Koundrioukoff & Michelle Debatisse

  4. Institut Curie, UMR3244 (CNRS), 26 rue d'Ulm, Paris, 75005, France

    Alain Nicolas

Authors
  1. Hervé Técher
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  2. Stéphane Koundrioukoff
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  3. Alain Nicolas
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  4. Michelle Debatisse
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Corresponding author

Correspondence to Michelle Debatisse.

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PowerPoint slides

Glossary

Replication stress

In this Review, defined as a condition that impedes the progression of replication forks strongly enough to prevent the maintenance of the normal replication rate through an increase in the density of the initiation event.

Replication origins

Chromosome regions from which replication starts, giving rise to bidirectional replication forks.

Topologically associated domains

(TADs). Sub-megabase-scale DNA domains comprising sequences that physically interact with each other more frequently than with regions located in adjacent domains. TADs represent spatially segregated genome regions.

Origin recognition complex

(ORC). A hetero-hexameric complex composed of six subunits (ORC1–6) that binds the DNA or the chromatin, which is a prerequisite for the subsequent building of functional replication origins.

DNA combing

A method stretching single DNA molecules on microscope coverslips, allowing their observation. In combination with in vivo labelling of newly synthesized DNA with analogues of DNA precursors, it allows the study of DNA replication.

Triplex DNA

A single-stranded DNA region bound to the major groove of the DNA duplex forming a three-stranded helix, which normally occurs at sequences with mirror symmetry.

Hairpins

DNA structures in which a strand folds on itself and forms intra-strand base pairing.

G-Quadruplexes

Four repeats of at least three guanines that can interact to form four-stranded DNA structures.

Nucleotide excision repair

(NER). Repair pathway that removes a broad range of lesions that distort the DNA double-helix structure. Processing relies on XPF–ERCC1 and XPG, two structure-dependent nucleases that remove a short oligonucleotide containing the lesions. The gap is then filled and repair completed.

Replication protein A

(RPA). This heterotrimeric complex specifically binds single-stranded DNA and protects it from degradation. RPA is a key factor in fork protection, ATR-CHK1 checkpoint activation, and DNA repair.

γH2AX

Phosphorylation of histone H2AX on serine 139 (γH2AX) by DNA damage response (DDR) apical kinases (ATM, ATR and DNAPK) constitutes a general marker of DNA damage and replication stress.

Synthetic lethality

Lethal phenotype that is associated with the co-inactivation of two proteins, whereas inactivation of either one of the proteins alone is viable. It is an important strategy for developing new cancer therapies.

Isolation of proteins on nascent DNA

(iPOND). A method that enables the purification of newly synthesized DNA and associated proteins upon immunoprecipitation of nucleotides analogues incorporated at the fork during a short pulse-labelling period.

Senescence

A phenomenon by which cells stop proliferating. It is usually induced by shortening of the telomeres and subsequent DNA damage response (DDR) activation.

MUS81–EME1/2

Structure-dependent nuclease that efficiently processes various DNA structures containing a junction between single-stranded DNA and double-stranded DNA, including replication forks. MUS81 is the catalytic subunit and EME1 and EME2 are two regulatory subunits that peak in G2/M and S phase cells, respectively.

MRE11

Protein belonging to a complex comprising MRE11, RAD50 and NBS1 (the MRN complex). The MRN complex is recruited to double-strand breaks (DSBs), where it has crucial roles in damage repair, and to replication forks upon replication stress. MRE11 has 3′–5′ exonuclease and single-stranded DNA (ssDNA) endonuclease activities that together contribute to DSB resection by producing the 3′ ssDNA overhang required in homology-directed repair and checkpoint activation.

p53 binding protein 1

(53BP1). Recruited to double-strand break (DSB) sites, where it promotes end-joining and inhibits homologous recombination repair. Large 53BP1 bodies are also formed upon replication stress, supposedly protecting DSBs from extensive degradation.

Break-induced replication

(BIR). A type of homologous recombination-driven replication that extends one-ended DNA double-strand breaks, which can form upon fork collapse. New DNA synthesis begins when the 3′ end of a break invades a double-strand template, forming a moving D-loop. Then, the second strand is synthesized.

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Técher, H., Koundrioukoff, S., Nicolas, A. et al. The impact of replication stress on replication dynamics and DNA damage in vertebrate cells. Nat Rev Genet 18, 535–550 (2017). https://doi.org/10.1038/nrg.2017.46

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