The structural basis of DDK-dependent replicative helicase activation

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.

During eukaryotic DNA replication, the replicative helicase MCM2-7 becomes loaded onto dsDNA as an inactive double hexamer and serves as the platform for replisome assembly. To initiate helicase activation, DDK kinase binds to and phosphorylates the MCM2-7 double hexamer, which in turn allows the recruitment of several helicase activation factors. This reaction is highly specific, as DDK fails to phosphorylate the MCM2-7 single hexamer. DDK and the associated helicase activation factors are expressed in cells at low levels and their recruitment to the MCM2-7 double hexamer is the rate limiting step in DNA replication. Consistently, the efficient and regulated helicase activation is essential for accurate DNA replication, but also very important for genome stability, stem cell homeostasis, aging and tumorigenesis. However, the structural basis of how DDK recognises the MCM2-7 double hexamer and how activation factors subsequently bind to MCM2-7 is completely unknown. Our overall aim is to use single-particle cryo-electron microscopy to fully elucidate the structures of the DDK-dependent helicase activation intermediates at an atomic level. We will reconstitute in a stepwise manner DDK-dependent helicase activation employing a recently developed protocol that supports efficient complex assembly for subsequent structural analysis. This research will provide fundamental mechanistic insights into the DDK-dependent phosphorylation of the MCM2-7 double hexamer and the following complex assembly pathway. Importantly, our study will serve not only as a foundation for subsequent helicase activation steps, but also a paradigm for phosphorylation of DDK substrates in replication checkpoint signalling, DNA repair, DNA recombination and chromosome segregation. Furthermore, the structural knowledge could be important for the development of small molecule inhibitors of DNA replication.

The main groups that will be impacted by the proposed research include:

1) Academic sector.
The scientific community will be the main immediate beneficiary as the proposed research will provide new knowledge and structures of the eukaryotic MCM2-7 helicase bound to activation factors. A detailed insight into the mechanism of helicase activation will have an impact on a broad range of researchers in the academic, clinical and agricultural fields since we will be able to further add to the current understanding of the fundamental process of DNA replication and its control, which is very important for stem cell function, seed development, aging and cancer. The project will provide a clear opportunity for career development and training for the PDRAs, which will be supported by the PI and the outstanding postdoctoral development centre of Imperial College. The project will benefit the UK economy and society in the short term by providing employment and in the long term by training the PDRAs to a high level and offering experience in public engagement. The researchers (postdocs and associated Master students, PhD students and technician) carrying out the project will gain invaluable experience in state-of-the-art biochemistry of molecular machines, cryo-EM sample preparation and modelling of structural data. Our research environment is fully set up to tackle the challenges of this project and uses an interdisciplinary approach that will only enhance the training of the PDRAs by adding to their ability to work with other research groups and UK biotechnology and pharmaceutical companies.

2) Industry sector.
The proposed structural work on helicase activation will benefit the pharmaceutical, healthcare and agricultural sector in the UK by providing detailed information of the function of the relevant factors and associated structures, which will support the development of helicase activation inhibitors as novel anti-fungal agent with applications in agriculture and medicine and in the long-term as an anti-cancer therapeutic. The basic research into cellular biology is generally not covered by these sectors and so our work will provide the fundamental basis on which translation research is built upon. With the support of the Imperial College London Centre for Drug Discovery Science and Imperial Corporate Partnerships and the Enterprise teams we will be able to exploit any new knowledge or development of a new application. Indeed, just recently the Molecular Sciences Research Hub with associated start-up companies opened in close proximity at the new White City campus. CS already met with several of the chemical-biology groups to evaluate the best strategies to develop novel inhibitors with potential for shared PhD studentships to carry out collaborative translational work.

3) Government.
The design of marketable inhibitors against helicase activation, with the support of Imperial College London Centre for Drug Discovery Science (CDDS), will be beneficial to the UK government. The inhibitors will help alleviate the current high pressure on the healthcare industry for novel anti-fungal medicine and help the agricultural industry increase plant crop yield and in turn will help with financial growth.

4) Society.
We believe it is important to increase scientific and health literacy. We will therefore put a great deal of effort into communicating our findings and methodologies to the general public. This will include: displaying our findings on public websites, use of social media, development of educational material, participation in Imperial College London organised public engagement events, two workshops (including the "Little People UK" as representatives of Meier-Gorlin syndrome patients) and the design of a publicly accessible video.