Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox

To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.European Research Council (ERC-2014-CoG 647344

The E1A-Associated p400 Protein Modulates Cell Fate Decisions by the Regulation of ROS Homeostasis

The p400 E1A-associated protein, which mediates H2A.Z incorporation at specific promoters, plays a major role in cell fate decisions: it promotes cell cycle progression and inhibits induction of apoptosis or senescence. Here, we show that p400 expression is required for the correct control of ROS metabolism. Depletion of p400 indeed increases intracellular ROS levels and causes the appearance of DNA damage, indicating that p400 maintains oxidative stress below a threshold at which DNA damages occur. Suppression of the DNA damage response using a siRNA against ATM inhibits the effects of p400 on cell cycle progression, apoptosis, or senescence, demonstrating the importance of ATM–dependent DDR pathways in cell fates control by p400. Finally, we show that these effects of p400 are dependent on direct transcriptional regulation of specific promoters and may also involve a positive feedback loop between oxidative stress and DNA breaks since we found that persistent DNA breaks are sufficient to increase ROS levels. Altogether, our results uncover an unexpected link between p400 and ROS metabolism and allow deciphering the molecular mechanisms largely responsible for cell proliferation control by p400

Cohesin Protects Genes against γH2AX Induced by DNA Double-Strand Breaks

Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome

m6A RNA modification as a new player in R-loop regulation

International audienceNo abstract availabl

The Secret Life of Chromosome Loops upon DNA Double-Strand Break

International audienc

A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks

International audienc

Régulation de l'histone acétyltransferase TIP60

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Taming Tricky DSBs: ATM on duty

International audienceAtaxia Telangiectasia Mutated (ATM) has been known for decades as the main kinase mediating the DNA Double-Strand Break Response (DDR). Extensive studies have revealed its dual role in locally promoting detection and repair of DSBs as well as in activating global DNA damage checkpoints. However, recent studies pinpoint additional unanticipated functions for ATM in modifying both the local chromatin landscape and the global chromosome organization, more particularly at persistent breaks. Given the emergence of a novel and unexpected class of DSBs prevalently arising in transcriptionally active genes and intrinsically difficult to repair, a specific role of ATM at refractory DSBs could be an important and so far overlooked feature of Ataxia Telangiectasia (A-T) a severe disorder associated with ATM mutations

Non-canonical DNA/RNA structures during Transcription-Coupled Double-Strand Break Repair: Roadblocks or Bona fide repair intermediates?

International audienc

Quantitation of DNA double-strand break resection intermediates in human cells

International audience5' strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5' strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle-dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection