Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

Tolerance of DNA replication stress – TODS

The DNA Damage Response and Cancer

Aberrant entry into S phase yields replication associated double-strand DNA breaks. Many chemotherapies aim at selective killing of tumour cells via stress overload. Hence, a thorough understanding of the mechanisms of the DNA damage response is necessary to improve current cancer treatments.

Replication associated mechanisms of DNA damage signalling and repair

1. We aim at reconstituting in vitro signalling cascades implicated in the response to DNA replication stress. We wish to dissect protein assemblies implicated in DNA damage signalling and to unveil key structural features of damaged DNA that triggers the assembly of DNA damage signalling complexes.<br />2. We aim at identifying novel DNA damage response factors that play a key role in tolerance of DNA replication stress<br />3. We explore how replication dynamics is regulated in response to DNA replication stress<br /><br />This work should yield novel prognostic and diagnostic tools as well as new therapeutic targets for the treatment of cancer

To gain new insights into the factors and mechanisms implicated in the cellular response to DNA replication stress, we have developed a cell free system competent for DNA damage induced signalling.
To uncover key players of the cellular response to replication blocks, we have developed a novel procedure for the purification and identification of proteins associated with newly synthesised DNA near replication forks.
To dissect he composition of molecular machines recruited to chromatin in response to DNA damage, we are purifying chromatin bound complexes in native conditions by size exclusion and affinity chromatography.
The function of key players of the DNA damage response is probed using a variety of complementary biochemical and cell biological approaches, including cutting edge techniques, such as molecular combing, which permits the visualisation of replication tracts a the single molecule level.

1. We have identified a specific damaged DNA substrate that can induced DNA damage signalling in vitro, and we have identified several of the factors that are implicated in this signalling reaction. Our current focus is on how phosphorylation of key factors relay to effector proteins implicated in DNA repair.
2. We have identified novel factors that are specifically recruited in response to a replication block induced by camptothecin, a replication inhibitor.
3. Proteins implicated in Fanconi anaemia play a key role in the cellular response to DNA replication stress and in the maintenance of genome stability. We found how two key FA factors, FANCD2 and FANCI, translocate to chromatin and control DNA replication under stressful conditions via direct association with components of the replisome.

The DNA damage signalling cell free system should facilitate the screening of small molecule modulators of DNA damage signalling.
The identification of novel factors recruited to replication forks stalled by treatment with camptothecin should provide new functional biomarkers for the treatment of colon cancer.

Our recent data have not yet been submitted for publication but have been presented at international and national meetings. We plan to submit the first part of this work for publication at the end of 2012. So far, we have published an invited review on the role of Fanconi anemia proteins in the response to replication stress.
Publication: Constantinou A (2012). Rescue of replication failure by Fanconi anaemia proteins. Chromosoma, 121, 21-36.
Congrès, présentation orale: Experimental Biology 2012. San Diego Convention Center, San Diego, CA, USA, April 21-25. Posters: a) Responses to DNA damage: From Molecular Mechanisms to Human Disease, 2011, Egmond aan Zee, The Netherlands, April 3-8. b) 9ème Colloque des 3 Rs, 2011, presqu’île de Giens, France, 2 - 5 Mai. c) 3nd Workshop de l’axe 2 Génome Structure et Fonctions 2012, Toulouse, France, 5 - 6 avril.

Our research goal is to understand how cells respond to stress during DNA replication, in relation to human disease. In S phase, the DNA damage response orchestrates and coordinates the repair of DNA lesions, and the resolution of problems during DNA replication, with ongoing cellular physiology. This response is notably implemented by the master checkpoint kinase ATM (which signals double-strand breaks) and ATR ( which signals blocked replication forks), together with a number of sensors, transducers, and effectors proteins. Failure to correctly sense and/or repair DNA damage is the underlying cause of a number of human disease, that can manifest clinically as neurological defects, immunodeficiency, developmental defects, premature ageing and cancer predisposition. Fanconi anaemia is an inherited cancer prone disorder cause by defects in the DNA damage response. To date, 13 FA genes have been discovered. Accumulating evidence suggests that FANC proteins represent a critical node in an integrated network of DNA damage response proteins, which provides the cell with the capacity to coordinate the action of molecular machines involved in the detection and in the signaling of DNA lesions, in DNA repair, in DNA replication and in the regulation of cell cycle progression and cell survival. At a molecular level, however, the roles of Fanconi anemia proteins remain largely elusive.

Our objective is to understand: a) how pathological DNA replication structures are sensed, signaled and processed; b) how DNA damage signaling is relayed to effector proteins and the role of Fanconi anaemia proteins in these processes; and c) how the Fanconi anaemia protein FANCD2 promotes DNA replication under stressful conditions. To achieve this, we will develop new systems to recapitulate DNA damage signaling and DNA repair in human cell extracts, and we will use a combination of biochemical, chromatin immunoprecipitation and molecular combing approaches to dissect the role of FANCD2 in replication dynamics.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 400,000 euros
Beginning and duration of the scientific project: - 48 Months

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