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

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

2005

• Lambert S, Carr AM.
  Checkpoint responses to replication fork barriers
  Biochimie, 87(7):591-602
• 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

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