The structural basis of DDK-dependent replicative helicase activation

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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.