Molecular mechanisms and physiological functions of DNA damage condensates

DNA损伤凝聚物的分子机制和生理功能

• 批准号: 419138288
• 负责人: Professor Dr. Simon Alberti
• 金额: --
• 依托单位: Biotechnologisches Zentrum (Biotec)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/419138288?language=en
• 关键词: Molecular; mechanisms; physiological; functions; DNA

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.

DNA双链断裂（DSB）是一种有害的DNA损伤类型，可通过DNA修复途径抵消。细胞内的遗传和药理学操作已经产生了DSB修复的细胞参与者和事件的全面画面。然而，我们对这些重要修复过程的分子和机制的理解仍然有限，并且在试管中研究DSB修复的尝试是基于简单的系统，这些系统没有重现在细胞中观察到的DNA损伤位点的出现。因此，对DSB修复机制的全面理解仍然缺失。我们聚焦于关键的DNA损伤酶聚（ADP）核糖聚合酶（PARP1），已经能够在试管中构建早期活性DSB缩合物。这些凝聚物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，机械解剖的DNA损伤识别和通路决策的分子事件。这个建议的目标是提供一个详细的分子和介观尺度的理解DSB修复的早期步骤。为了促进DNA双链断裂的修复，两个DNA末端必须保持接近。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末端保持邻近，仍然是个谜。使用我们自下而上的方法来重建DNA损伤位点，我们已经能够证明PARP1分子聚集在DNA损伤周围。这些PARP 1簇阻止DNA末端的分离，并促进修复酶的募集。破译这个复杂的装配需要一个多学科的方法相结合的重组生物化学，分子生物物理学和单分子的方法。该联盟结合了两个小组的不同专业知识，以全面表征在突触DNA末端组装的DSB缩合物。我们的目标是揭示DSB冷凝物组装的调控原理，并为这些冷凝物在细胞中的功能和疾病相关作用提供重要的机制见解。