Molecular mechanisms and physiological functions of DNA damage condensates

DNA双链断裂(DSB)是一种有害的DNA损伤类型，DNA修复途径可以抵消这种损伤。细胞内的遗传和药物操纵已经产生了细胞参与者和DSB修复事件的全面画面。然而，我们对这些重要修复过程背后的分子和机制的了解仍然有限，在试管中研究DSB修复的尝试是基于简单的系统，而不是像在细胞中观察到的那样概括DNA损伤位置的出现。因此，对DSB修复的全面机制仍缺乏了解。聚焦于关键的DNA损伤酶多聚(ADP)核糖聚合酶(PARP1)，我们已经能够在试管中构建早期活性的DSB缩合物。这些凝析油的稳定性取决于多种成分，需要持续的能量输入，因此概括了细胞中的关键观察结果。使用这个系统，我们现在处于一个独特的位置，可以机械地剖析DNA损伤识别和通路决策的分子事件。这项提议的目标是提供对DSB修复早期步骤的详细的分子和中尺度理解。为了促进DNA双链断裂的修复，DNA两端必须保持接近。这一步至关重要，因为DSB修复的所有下游步骤都依赖于它。细胞如何确保断裂的DNA末端保持在附近，仍然是个谜。使用我们的自下而上的方法重建DNA损伤部位，我们已经能够证明PARP1分子聚集在DNA损伤周围。这些PARP1簇防止DNA末端分离，并促进修复酶的招募。破译这种复杂的组装需要结合重组生物化学、分子生物物理学和单分子方法的多学科方法。该联盟结合了两个小组的不同专业知识，以全面表征聚集在突触DNA末端的DSB凝聚体。我们的目标是揭示DSB冷凝物组装的调节原理，并为这些冷凝物在细胞中的功能和疾病相关角色提供重要的机械见解。

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.