Regulation of blocked-DSB repair by DNA-PKcs and ATM kinases

Resumen

Given the important threat to genome integrity that double strand breaks (DSBs) pose, understanding the molecular mechanisms that govern DSB repair is extremely relevant. Notably, the structures that are present at DSB ends define their complexity and are considered putative determinants for repair pathway choice and outcome. This question, however, has not been sufficiently elucidated due to the difficulty to induce homogeneous populations of DSBs with defined end structures. Taking the advantage of a recently developed genetic strategy to induce populations of DSBs that are homogeneous in end-structure, we have dissected pathways specifically required to repair blocked DNA ends during G0/G1 stages of the cell cycle. For this, we have performed CRISPR/Cas9 genetic screens in human cells and candidates identified have been characterised, together with additional related factors. We have found that there is an established preference for the repair of TOP2-DSBs by the unblocking activity of TDP2 instead of end-processing pathways mediated by nucleases, which are only necessary when the ends are irreversibly blocked. This hierarchy contributes to ensure genome stability and is disrupted in the absence of DNA-PKcs. We also demonstrate that the role of ATM in blocked DSB repair is mainly related with the nucleolytic pathway, although it could also protect the ends from an excessive processing. Furthermore, we show that the established hierarchy that prioritise TDP2 activity to repair TOP2-DSBs avoids malignant transformation and cancer development.