Molecular mechanisms and physiological functions of DNA damage condensates

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DNA double strand breaks (DSBs) are a detrimental type of DNA damage, which is counteracted by DNA repair pathways. Genetic and pharmacological manipulations inside cells have generated a comprehensive picture of the cellular players and events of DSB repair. However, our molecular and mechanistic understanding underlying these vital repair processes remains limited and attempts to investigate DSB repair in the test tube were based on simple systems that did not recapitulate the emergence of DNA damage sites as observed in cells. Accordingly, a comprehensive mechanistic understanding of DSBs repair is still missing.Focussing on the key DNA damage enzyme poly(ADP) ribose polymerase (PARP1), we have been able to build early active DSB condensates in the test tube. The stability of these condensates depends on multiple components and requires a continuous input of energy and thus recapitulates key observations in cells. Using this system, we are now in a unique position to mechanistically dissect the molecular events underlying DNA damage recognition and pathway decision making.The goal of this proposal is to provide a detailed molecular and mesoscale understanding of the early steps in DSB repair. To facilitate the repair of DNA double strand breaks, both DNA ends must stay in proximity. This step is essential because all downstream steps of DSB repair depend on it. How cells ensure that broken DNA ends remain in proximity, remains enigmatic. Using our bottom-up approach to reconstitute DNA damage sites, we have been able to demonstrate that PARP1 molecules cluster around DNA lesions. These PARP1 clusters prevent the separation of DNA ends and facilitate recruitment of repair enzymes. Deciphering this complex assembly requires a multidisciplinary approach combining reconstitution biochemistry, molecular biophysics and single-molecule approaches. This consortium combines the diverse expertise of two groups to comprehensively characterize DSB condensates that assemble on synapsed DNA ends. We aim to reveal the regulatory principles underlying DSB condensate assembly and provide important mechanistic insights into functional and disease-associated roles of these condensates in cells.