Understanding the molecular basis of checkpoint response during DNA double-strand break repair

了解 DNA 双链断裂修复过程中检查点反应的分子基础

• 批准号: MR/Y001192/1
• 负责人: Xiaodong Zhang
• 金额: $ 259.76万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FY001192%2F1
• 关键词: Understanding; molecular; basis; checkpoint; response

Our genomic information is stored in DNA, which has a double helical structure and is organised into nucleosomes and chromosome. However our DNA suffers from constant assaults from both external and internal sources such as UV/ionizing radiation, chemicals from smoke and drugs as well as molecules produced from our body's normal metabolic activities. Some of the assaults lead to severe DNA damages such as a double strand break (DSB), when both DNA strands are broken and therefore cells can't use the intact strand as a template for repair. If unrepaired or misrepaired, a DSB can result in changes in our DNA that lead to cell death or permanent changes in our genes. These factors contribute to aging and other human diseases such as cancer. Fortunately our cells have developed several ways to repair DSBs, especially to ensure they are repaired before our cells duplicate and pass our genes to next generation of cells. We want to study how this is achieved. Currently we know that three very large protein molecules called DNA-PKcs, ATM and ATR are the master coordinators. They modify other protein molecules (phosphorylate them) to enable them to carry out repair and to ensure the cells are slowed down or stopped progressing to the next stage, until the repair is complete. How precisely these master coordinators carry out these complex tasks is currently unknown. We plan to study ATM and ATR, both are involved in the process that carries out DNA repair faithfully. We want to find out how they are recruited to the damaged site (in the context of nucleosomes and chromosome), how they are activated by other factors and how they then modify other protein substrates that lead to slow down or halt cell cycle progession. We will use purified molecules to assemble these complexes and to study them using cutting edge methodolgies such as cryo electron microscopy, which allow us to obtain high resolution 3-dimensional structures of molecules and their complexes. These structures, complemented by biochemical, biophysical and cellular studies, will inform us the molecular details on how they are recruited to a damaged DNA within the chromosome, how other factors (activators) change their structrues to enable them to modify their substrates and finally how they act on their substrates efficiently, especially some of them are distally located. Our work will provide crucial information on these important large molecules, fill in a critical knowledge gap on how our cell cycle progress is controlled upon a DSB, can have profound therapeutic implications. Both proteins are tumor suppressors and mutations are found in cancer patients so our mechanistic understanding will provide molecular explanation for how these mutations increase cancer development. Further, these proteins are validated drug targets and our knowledge will help with new avenues for future drug development.

我们的基因组信息存储在 DNA 中，DNA 具有双螺旋结构，并组织成核小体和染色体。然而，我们的 DNA 不断受到外部和内部来源的攻击，例如紫外线/电离辐射、烟雾和药物中的化学物质以及我们身体正常代谢活动产生的分子。一些攻击会导致严重的 DNA 损伤，例如双链断裂 (DSB)，此时两条 DNA 链均断裂，因此细胞无法使用完整的链作为修复模板。如果未修复或修复不当，DSB 可能会导致我们的 DNA 发生变化，从而导致细胞死亡或基因发生永久性变化。这些因素导致衰老和癌症等其他人类疾病。幸运的是，我们的细胞已经开发出多种修复 DSB 的方法，特别是确保它们在我们的细胞复制并将我们的基因传递给下一代细胞之前得到修复。我们想研究这是如何实现的。目前我们知道，DNA-PKcs、ATM 和 ATR 三种非常大的蛋白质分子是主协调员。它们修饰其他蛋白质分子（将其磷酸化），使它们能够进行修复，并确保细胞减慢或停止进入下一阶段，直到修复完成。目前尚不清楚这些主协调员如何精确地执行这些复杂的任务。我们计划研究ATM和ATR，两者都参与了DNA忠实修复的过程。我们想了解它们如何被招募到受损位点（在核小体和染色体的情况下），它们如何被其他因素激活，以及它们如何修饰其他蛋白质底物，从而导致细胞周期进展减慢或停止。我们将使用纯化的分子来组装这些复合物，并使用冷冻电子显微镜等尖端方法来研究它们，这使我们能够获得分子及其复合物的高分辨率3维结构。这些结构，辅之以生物化学、生物物理和细胞研究，将告诉我们分子细节：它们如何被招募到染色体内受损的DNA上，其他因子（激活剂）如何改变它们的结构，使它们能够修改它们的底物，以及最后它们如何有效地作用于它们的底物，特别是其中一些位于远端的底物。我们的工作将提供有关这些重要大分子的重要信息，填补关于如何通过 DSB 控制细胞周期进程的关键知识空白，可能具有深远的治疗意义。这两种蛋白质都是肿瘤抑制因子，并且在癌症患者中发现了突变，因此我们的机制理解将为这些突变如何促进癌症发展提供分子解释。此外，这些蛋白质是经过验证的药物靶点，我们的知识将有助于为未来药物开发提供新途径。