Cell response to replication stress

Keywords: DNA replication, Homologous recombination, S. pombe yeast, Genetic instability, DNA Integrity Checkpoints

Consultez le Read the scientific activity report.. (pdf 370Ko, last update 19th, january 2010)

Chromosome replication occurs during the S phase of the cell cycle and is initiated by multiple origins that are activated at the beginning, in the middle or at the end of the S phase. Each replication fork must replicate several tens of thousands of bases before meeting a converging fork. During this DNA synthesis, the progression of replication forks may be compromised by Replication Fork Barriers (RFBs) that slow down, arrest or stall the forks. RFBs have various origins, which can be for example secondary DNA structures, the presence of tightly anchored DNA-protein complexes, or DNA damage. These obstacles are a threat to the integrity of replication forks and a potential source of genome instability. Confronted with this risk, cells have developed fundamental mechanisms to be able to faithfully transmit hereditary material when replication is hindered (Fig. 1).

Fig. 1 

Among these mechanisms, genome integrity checkpoints and homologous recombination are essential mechanisms that enable to maintain genomic stability when problems occur during replication. However, these mechanisms can also be double-edged swords, that help to maintain the continuity of DNA synthesis when replication is compromised, but at the expense of genetic instability. This raises regulation issues for the mechanisms involved during replication. Our team is attempting to understand molecular transactions controlling genome stability when DNA replication is perturbed. To investigate these issues, we use Schizosaccharomyces pombe yeast as a model organism that enables to combine genetic approaches and molecular and cellular approaches (Immuno-precipitation of chromatin associated proteins, replication intermediates analysis, pulse-field gel electrophoresis, in vivo microscopy).

Forks progression can be compromised by various drugs: hydroxyurea that results in nucleotide pool depletion and forks slowing down, or genotoxic agents inducing DNA damages. We have recently brought to evidence that the DNA replication checkpoint protects the genome from chromosome breakages during S phase in response to HU-induced stalled forks. We proposed that chromomes breackage result from cleavage of unstabilized stalled forks by the endonuclease M us81 (Fig. 2). In addition, Mus81-dependent forks cleavage is facilitated by the helicase Rqh1 (Froget et al. 2008 Mol. Biol. Cell).

Fig. 2 

We dispose of a genetic assay to induce stalling of a single replication fork with a protein/DNA complex. This system can mimic natural replication arrests that occur during every replication and allow to investigate the behavior of such arrested fork in vivo. When the We previously reported that when fork arrest is unduced, recombination pathway becomes necessary for cell survival and that such replication fork stalling is a hot spot for recombination and chromosomal rearrangements (Lambert et al. 2005 Cell). Hence, this system is an appropriate tool to understand the mode of action of homologous recombination in the processing of stalled replication forks (Fig. 3).

Fig. 3 

We explore two complementary aspects using these tools:

• Identification of factors and molecular mechanisms that maintain genome stability in response to replication perturbations.
  
• Orchestration of recombination actors on stalled or slowed-down replication forks.

Alteration of the replication dynamic appears to be linked to tumor progression in humans. On one hand, oncogene-induced proliferation leads to aberrant DNA replication which in turn can be at the origin of genome instability during early stages of tumor development. Genome integrity checkpoints thus act as a biological barrier to tumor development. However, the molecular mechanisms linking resplicative stress to genome instibility remain poorly understood. On the other hand, some genetic syndromes associated to tumor predisposition, such as the Bloom syndrome, are characterized by defect in proper response to replicative stress. Thus, our basic research that focuses on how cells deal with replicative stress to limit genome instability, helps understanding the mechanisms that bring a normal cell into a tumoral state. In addition, many genes involved in maintaining genome stability in yeasts are involved in tumor suppressor pathways in humans. It is thus possible to use a simple model organism to dissect the fundamental role of homologs of tumor suppressors in maintaining genome stability in response to replication perturbations. This is the case in particular for the BLM homolog helicase Rqh1, which leads to Bloom syndrome when absent.

Last update: January 2010

Key publications

2009

• Mizuno K, Lambert S, Baldacci G, Murray JM, Carr AM
  Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism
  Genes Dev., 23(24):2876-86 - Abstract
  Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage-fusion-bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.
  - Full version

2008

• Froget B, Blaisonneau J, Lambert S *, Baldacci G. (*corresponding auteur)
  Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint
  Mol Biol Cell., 19(2):445-56 - Abstract
  During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.
  - Full version

2007

• Delacote F, Deriano L, Lambert S, Bertrand P, Saintigny Y, Lopez BS.
  Chronic exposure to sublethal doses of radiation mimetic Zeocin selects for clones deficient in homologous recombination
  Mutat Res., 615(1-2):125-33
• Lambert S, Froget B, Carr AM.
  Arrested replication fork processing: interplay between checkpoints and recombination
  DNA Repair (Amst), 6(7):1042-61 - Abstract
  The arrest of DNA replication by DNA damage, nucleotide depletion, DNA-protein complexes or following clashes between transcription and replication factors all have the capacity to promote genome instability. In this review, we discuss how DNA replication is regulated by the checkpoint pathways that stabilise arrested replication forks and the recombination factors that process specific DNA structures resulting from fork arrest. We examine what is known about the interplay between the checkpoints and the recombination apparatus and review the evidence for a recombination-based fork restart pathway in eukaryotes.

2005

• Lambert S, Carr AM.
  Checkpoint responses to replication fork barriers
  Biochimie, 87(7):591-602 - Abstract
  The fidelity of DNA replication is of paramount importance to the maintenance of genome integrity. When an active replication fork is perturbed, multiple cellular pathways are recruited to stabilize the replication apparatus and to help to bypass or correct the causative problem. However, if the problem is not corrected, the fork may collapse, exposing free DNA ends to potentially inappropriate processing. In prokaryotes, replication fork collapse promotes the activity of recombination proteins to restore a replication fork. Recent work has demonstrated that recombination is also intimately linked to replication in eukaryotic cells, and that recombination proteins are recruited to collapsed, but not stalled, replication forks. In this review we discuss the different types of potential replication fork barriers (RFB) and how these distinct RFBs can result in different DNA structures at the stalled replication fork. The DNA structure checkpoints which act within S phase respond to different RFBs in different ways and we thus discuss the processes that are controlled by the DNA replication checkpoints, paying particular attention to the function of the intra-S phase checkpoint that stabilises the stalled fork.
• Lambert S, Watson A, Sheedy DM, Martin B, Carr AM.
  Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier
  Cell., 121(5):689-702 - Abstract
  Genomic rearrangements linked to aberrant recombination are associated with cancer and human genetic diseases. Such recombination has indirectly been linked to replication fork stalling. Using fission yeast, we have developed a genetic system to block replication forks at nonhistone/DNA complexes located at a specific euchromatic site. We demonstrate that stalled replication forks lead to elevated intrachromosomal and ectopic recombination promoting site-specific gross chromosomal rearrangements. We show that recombination is required to promote cell viability when forks are stalled, that recombination proteins associate with sites of fork stalling, and that recombination participates in deleterious site-specific chromosomal rearrangements. Thus, recombination is a "double-edged sword," preventing cell death when the replisome disassembles at the expense of genetic stability.

2003

• Lambert S, Mason SJ, Barber LJ, Hartley JA, Pearce JA, Carr AM, McHugh PJ
  Schizosaccharomyces pombe checkpoint response to DNA interstrand cross-links
  Mol Cell Biol., 23(13):4728-37