Genome duplication defects caused by homologous recombination deficiency. – StressHoR

WP1) The cellular responses and consequences of endogenous replication stress
This WP covers molecular mechanisms of replication restart and fork protection by HR in fission yeast, the proteomics and genomic of DNA Damage Foci in mammalian cells defective for HR, the characterization of a novel factor, SAMHD1, in the processing of replication forks in mammals, the proteomics of stressed replication forks in mammals and yeast.
WP2) The origin of endogenous replication stress revealed by HR deficiency.
This WP covers the nature of replication stress in HR deficient cells in mammals, The characterization of fission yeast rad51 separation of function mutants.

A new mechanism linking replication stress to inflammation : An unexpected result obtained during the course of the characterization of the role of SAMHD1 at stalled forks is the fact that aberrant processing of arrested forks in the absence of SAMHD1 leads to the accumulation of cytosolic ssDNA.

Unprotected replication forks are converted into mitotic sister chromatid bridges: Recombination factors protect replication forks from degradation. Fission yeast Rad52/Rad51 protect forks in a recombination-independent manner. Unprotected forks challenge replication termination and are converted into mitotic sister chromatid bridges.

The non-homologous end joining factor Ku orchestrates replication fork resection and fine-tunes Rad51-mediated fork restart: Ku orchestrates a two-steps DNA end-resection of terminally-arrested and unbroken forks.

The oxydative stress is an important cause of endogenous replication stress: Surprisingly, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Therefore, the cellular redox status strongly impacts genome duplication and transmission.

Impact of the distance between double strand breaks and role of the cohesion complex in the S phase : DNA double-strand breaks (DSB) are very harmful lesions that can generate genome rearrangements. We propose that close ends are immediately/rapidly tethered and ligated, whereas distant ends first require synapsis of the distant DSEs prior to ligation. This «spatio-temporal« gap gives time and space for CtIP to initiate DNA resection, suggesting an involvement of single-stranded DNA tails for ECS capture. These data are particularly important with DSB generated by replication stress since this generates single-ended DSB; therefore end-joining of such DSB involved inevitably distant DSB.

This proposal will clarify the causal relationships between intrinsic replication stress and genetic instability, and should lead to the emergence of novel markers indicative of latent replication stress.

Magdalou I, Lopez BS, Pasero P. and Lambert SAE. (2014) The causes of replication stress and their consequences on genome stability and cell fate. Seminars Cell and Dev. Biol. 30, 154-164.
Menolfi D, Delamarre A, Lengronne A, Pasero P and Branzei D (2015) Essential roles of the Smc5/6 complex in replication through pausing sites and endogenous DNA damage tolerance. Mol Cell, 60, 835-46

Vindigni A and Pasero P (2017) Nucleases acting at stalled forks: how to reboot the replication program with a few shortcuts. Annual Review Genetics, 51. in press
Menolfi D, Delamarre A, Lengronne A, Pasero P and Branzei D (2015) Essential roles of the Smc5/6 complex in replication through pausing sites and endogenous DNA damage tolerance. Mol Cell, 60, 835-46

Ait Saada A, Teixeira-Silva A, Iraqui I, Costes A, Hardy J, Paoletti G, Fréon K, Lambert SAE. Unprotected replication forks are converted into mitotic sister chromatid bridges. Mol Cell. 2017 May

Gemble S, Buhagiar-Labarchede G, Onclercq-Delic R, Denis Biard, Lambert S, and Mounira Amor-Gueret . (2016). A balanced pyrimidine pool is required for optimal CHK1 activation to prevent ultrafine anaphase bridges formation. J. of Cell Science. 2016 Aug 15;129(16):3167-77.

Wilhelm T, Ragu S, Magdalou I, Machon C, Dardillac E, Técher H, Guitton J, Debatisse M and Lopez BS(2016). Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress. PLoS Genet., 12(5): e1006007. doi:10.1371/journal.pgen.1006007

Gelot C, Guirouilh-Barbat J, Le Guen T, Dardillac E, Chailleux C, Canitrot Y and Lopez BS (2016). The cohesin complex prevents the end-joining of distant DNA double-strand ends. Mol Cell, 61, 15-26.

Guirouilh-Barbat J, Gelot C, Xie A, Dardillac E, Scully R and Lopez BS (2016) 53BP1 Protects Against CtIP-dependent Capture of Ectopic Chromosomal Sequences at the Junction of Distant Double-Strand Breaks. PLoS Genet.

Failures in chromosome replication is a primary source of genetic instability, which ultimately leads to replication fork stalling, collapse or breakages and triggers the DNA Damage Response (DDR) activation. Impediments to fork progression induced by discrete obstacles, DNA damages or global replication stress generate genome instability. Impediments to fork progression might also challenge the completion of DNA replication and jeopardize chromosome segregation, resulting in mitosis defects and aneuploidy. Investigating the nature and the consequences of replication stress is of crucial importance to understand the relationships between defective fork progression and genome instability.
Replication stress activates the DDR which coordinates a network of pathways including DNA replication/repair/recombination, cell cycle checkpoints and chromosome segregation. The cascade of DDR activation and its components have been largely characterized in response to acute replication stress, but the cell response to chronic and endogenous replication stress remains poorly described, despite the fact that it represents the main source of replication-induced genetic instability. How cells adapt to chronic replication stress in order to complete DNA replication and protect their genome from deleterious rearrangements remains unknown.
Completion of DNA replication requires replication forks to be well-protected to ensure their stability/reactivation. Homologous Recombination (HR) plays a pivotal role in DNA repair and contributes to the robustness of DNA replication thank to its ability to repair replication-associated double strand breaks (DSBs) and to restart replication forks. Remarkably, HR-deficient cells display slower replication forks, mitosis defects and aneuploidy. Thus, cells defective for HR appear to suffer from intrinsic and chronic replication stress due to altered replication dynamic. Deciphering the biology of endogenous replication stress in the absence of HR should serve as a paradigm of how endogenous replication stress triggers genome instability and will provide novel insights into how cells adapt to chronic replication stress.
The overall objective of this proposal is to decipher the nature and the consequences of intrinsic replication stress resulting from HR defects. This proposal put forward a continuum of complementary studies associating the power of yeast molecular genetics to the cell biology of mammalian cells. Global methods of analysis will be employed to characterize: i) the impact of intrinsic replication stress on the dynamic of replication (genome-wide genomic approach), ii) the cell response to endogenous replication stress (Differential proteomic approach). In fission yeast, genetic assays will be employed to model the cell response and the consequences of defect in HR at a local replication stress site. The consortium brings together three teams (S. Lambert, B. Lopez and P. Pasero) and their know-how in mechanisms of HR, genome instability and cell response to replication stress.
In spite of its importance in genetic instability, little is known on the nature of endogenous replication stress, because it is in essence hard to reveal and analyze. One original aspect of the project is to use HR deficiency as a revelator of endogenous stress. This proposal will bring molecular insights in our understanding of the causal relationship between endogenous replication stress and genome instability, and the mechanisms by which HR contributes to the dynamic of replication. This proposal should lead to the emergence of novel markers indicative of latent replication stress, which are expected to be different from markers currently used to detect cellular response to massive replication stress. Novel markers of intrinsic replication stress markers will be useful for the detection of precancerous states and/or senescence and to investigate the effects of environmental factors on genetic instability on healthy tissues.