The structural basis of DDK-dependent replicative helicase activation

地球上所有生命的精确指令，关于它是如何形成和工作的，都包含在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遗传互补。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化的过程，涉及大量蛋白质在DNA上组装成一个高度复杂的机器。这台机器的成功组装确保了遗传信息的忠实复制。这台机器的一部分是核心的过程被称为DNA解旋酶。解旋酶的工作原理是与DNA结合并分离两条DNA链，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。在不利的情况下，解旋酶可能变得失调，导致作物植物的细胞应激、衰老、人类疾病或生长缺陷。已经有很多研究来理解环状DNA解旋酶如何首先结合到DNA上，并鉴定随后激活解旋酶进行DNA解旋的组分。目前，解旋酶活化所需的所有组分都是已知的，但不清楚它们如何起作用。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，我们将采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如何工作的信息，我们将能够观察解旋酶在与激活因子接触时所经历的任何变化。通过研究这些激活中间体中的多个，我们将能够生成一个解释整个过程的电影，因此将产生对DNA复制起始的基本见解。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的变化（突变），以破坏正常功能，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解旋酶激活机制的见解将产生对DNA最终解旋和DNA复制的基本步骤的概述。除了有助于对我们的细胞如何工作的基本理解外，这项研究对衰老、癌症等人类疾病、农业耕作具有重要意义，并有可能为医疗保健和农业部门设计新的特异性抑制剂。

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.