The structural basis of DDK-dependent replicative helicase activation

关于地球上所有生命如何形成和运作的准确指令包含在DNA中。为了生长、更新和繁殖细胞，必须首先复制它们的DNA，这样每个新的子细胞才能从母细胞获得完整的基因补充。只有当DNA复制完成后，细胞才能分裂。DNA复制是一个严格控制和高度组织的过程，它涉及大量蛋白质在DNA上组装成一个高度复杂的机器。这台机器的规范和成功组装确保了对遗传信息的忠实复制。在这个过程中，机器的一个核心部分被称为DNA解旋酶。解旋酶通过与DNA结合并分离两条DNA链来工作，以便为DNA复制机提供访问存储在每条DNA链中的遗传信息的途径。在不利的环境下，解旋酶可能变得失控，导致细胞应激、衰老、人类疾病或作物生长缺陷。已经有很多研究了解环状DNA解旋酶是如何与DNA结合的，并鉴定了随后激活解旋酶以解开DNA的成分。目前，解旋酶激活所需的所有成分都是已知的，但它们是如何发挥作用的还不清楚。我们的工作旨在揭示解旋酶被激活的详细机制。为了实现这一点，我们将使用高分辨率冷冻电子显微镜和复杂的计算方法来确定与激活因子结合的DNA解旋酶的3D形状。基本上，这将产生几个组件的蓝图。获得这些机器的蓝图将告诉我们很多关于它们是如何工作的，我们将能够观察到解旋酶在接触到激活因子时所经历的任何变化。通过研究这些激活中间体中的多个，我们将能够制作一部解释整个过程的电影，从而对DNA复制的启动产生基本的见解。为了验证对3D形状的正确解释，我们将在重要区域引入解旋酶(突变)的变化，以扰乱正常功能，并询问这些变化将如何影响细胞的正常功能。从长远来看，从这项研究和未来的工作中获得的对解旋酶激活的机械性洞察将产生对最终解开DNA和DNA复制的基本步骤的概述。除了有助于基本了解我们的细胞如何工作外，这项研究还对衰老、癌症等人类疾病、农业耕作有重要影响，并有可能导致为医疗保健和农业部门设计新的特定抑制剂。

The precise instructions of all life on earth, on how it is shaped and works is contained within DNA. In order to grow, renew and reproduce cells must first copy their DNA, so that each new daughter cell can receive the full genetic complement from the mother cell. It is only once DNA replication is complete that cell division can then occur. DNA replication is a tightly controlled and highly organised process, which involves a large number of proteins that assemble on DNA into a highly complex machine. The regulated and successful assembly of this machine ensures faithful copying of the genetic information. One part of the machine that is central to the process is called a DNA helicase. The helicase works by binding to DNA and separating the two DNA strands in order to provide the DNA copying machine with access to the genetic information that is stored within each DNA strand. Under unfavourable circumstances the helicase can become misregulated leading to cellular stress, aging, human disease or growth defects in crop plants. There has been much research into understanding how the ring-shaped DNA helicase first binds to DNA and into identification of the components that consequently activate the helicase for DNA unwinding. Currently, all components necessary for helicase activation are known, however it is not clear how they function. Our work aims to uncover the detailed mechanism by which the helicase is activated. In order to achieve this, we will employ the use of high-resolution cryo-electron microscopy and sophisticated computational methods to determine, the 3D shape of the DNA helicase bound to the activation factors. Basically, this will produce the blue-print of several components. Obtaining the blueprints of these machines will tell us a lot about how they work and we will be able to observe any changes the helicase undergoes when it comes into contact with the activation factors. By studying multiple of these activation intermediates we will be able to generate a movie that explains the overall process and therefore will yield fundamental insights into initiation of DNA replication. To verify the correct interpretation of the 3D shapes we will introduce changes to the helicase (mutations) at important regions in order to disrupt normal function and ask how these changes will affect the normal function of the cell. In the long-term, the mechanistic insight into helicase activation gained from this research and future work will generate an overview of the essential steps towards eventual unwinding of the DNA and DNA duplication. In addition to contributing to the basic understanding of how our cell works, this research has important implications for aging, human diseases such as cancer, agricultural farming and has the potential to lead to the design of new specific inhibitors for the healthcare and agricultural sector.