NEW APPROACHES TO STUDY DNA DOUBLE-STRAND BREAKS GENOME-WIDE AND IN SINGLE-CELLS

Cells have evolved several DNA repair mechanisms to maintain their genetic information unaltered and a DNA damage response pathway (DDR) coordinates DNA repair with several cellular events including a cell-cycle arrest until damaged DNA is repaired. When cells fail to repair DNA lesions, they undergo either apoptosis or cellular senescence. Tools commonly used to detect DNA lesions rely on indirect, antibody-based recognition of proteins associated to DNA breaks. Unfortunately, these tools do not allow direct and precise localization of the breaks, leaving several biological questions unanswered. I validated and optimized BLESS and BLISS, two methods that allow genome-wide single-nucleotide resolution mapping of DNA double strand-breaks (DSBs). Using these techniques, I studied the impact of DSBs on transcription. I characterized a DDR-dependent transcription inhibition around breaks. Differently, I observed that following macrophages LPS-stimulation, a transient wave of DSBs is induced at LPS-specific enhancers and it correlates with their transcription activation, thus suggesting a new mechanism for transcription activation involving controlled DNA damage generation. BLESS and BLISS cannot be applied to single-cell studies. Thus, I developed a new method, named DI-PLA, for the detection and imaging of DSBs in fixed cells and tissues. I applied DI-PLA to demonstrate that senescence cells and aged tissues accumulate DSBs, which are associated with persistent DDR activation, that is known to fuel cellular senescence. Finally, I developed a modification of BLESS to discriminate between DSB bearing telomeres and deprotected telomeres which could be applied to further characterize the mechanisms of DDR activation at telomeres.