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

我们的基因组信息储存在DNA中，DNA具有双螺旋结构，由核小体和染色体组成。然而，我们的DNA不断受到来自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，如双链断裂（DSB），当两条DNA链都断裂时，细胞不能使用完整的DNA链作为修复的模板。如果未修复或错误修复，DSB会导致我们的DNA发生变化，导致细胞死亡或我们的基因发生永久性变化。这些因素导致衰老和其他人类疾病，如癌症。幸运的是，我们的细胞已经发展出几种修复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。目前我们知道三个非常大的蛋白质分子，DNA-PKcs， ATM和ATR是主要的协调者。它们修饰其他蛋白质分子（使它们磷酸化），使它们能够进行修复，并确保细胞减缓或停止进入下一阶段，直到修复完成。这些主协调者如何精确地执行这些复杂的任务目前尚不清楚。我们计划研究ATM和ATR，它们都参与了忠实地进行DNA修复的过程。我们想知道它们是如何被招募到受损部位的（在核小体和染色体的背景下），它们是如何被其他因素激活的，以及它们是如何修饰其他蛋白质底物，从而减缓或停止细胞周期进程的。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三维结构。这些结构，辅以生物化学、生物物理和细胞研究，将告诉我们关于它们如何被招募到染色体内受损DNA的分子细节，其他因素（激活因子）如何改变它们的结构，使它们能够修饰它们的底物，最后它们如何有效地作用于底物，特别是其中一些位于远端。我们的工作将提供关于这些重要大分子的重要信息，填补关于如何在DSB上控制我们的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，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增加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的药物开发提供新的途径。

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.