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The structural basis of replicative helicase loading onto DNA

Reference	BB/S001387/1
Principal Investigator / Supervisor	Professor Christian Speck
Co-Investigators / Co-Supervisors
Institution	Imperial College London
Department	Institute of Clinical Sciences
Funding type	Research
Value (£)	453,833
Status	Completed
Type	Research Grant
Start date	26/11/2018
End date	25/05/2022
Duration	42 months

Abstract

During eukaryotic DNA replication, the replicative helicase MCM2-7 becomes loaded onto dsDNA to form a pre-replicative complex, which serves as the platform for replication fork assembly. Efficient and regulated helicase loading is essential for DNA replication, but has also high relevance for genome stability, stem cell homeostasis, aging and tumorigenesis. Structural changes from a spiral shaped MCM2-7 complex towards a perfectly shaped ring with DNA inserted represent the critical steps during the helicase loading, as this process physically links the enzyme to DNA. However, the underlying mechanisms of this reaction are completely unknown. Our overall aim is to use single-particle cryo-electron microscopy to fully elucidate how these structural changes and the DNA insertion reaction work at an atomic level. Our group developed highly efficient MCM2-7 in vitro assembly protocols and has a long track record of working at the interface of biochemistry and electron microscopy. Relying on our already obtained 3.9 Å structure of the related helicase loading complex, we will obtain the elusive structure of the helicase loading complex arrested before DNA insertion. Moreover, we aim to investigate the loading complex containing an essential factor of helicase loading, Cdt1, which has been modified in its interaction with either the helicase itself or the helicase loader, taking advantage of the knowledge from the recently solved helicase loading complex. Solving these structures will provide extensive mechanistic insights into the structural changes the eukaryotic helicase undergoes during the critical stages of helicase loading. Importantly, this work will serve as a paradigm for related processes involving regulated MCM2-7 ring opening/closing during DNA synthesis, DNA repair and potentially also DNA recombination, generating a wide interest. Furthermore, the structural knowledge could be important for the development of small molecule inhibitors of DNA replication.

Summary

The DNA in our cells and that of other lifeforms gives the precise instructions on how life is shaped and works. To grow, renew and reproduce, biological cells must first duplicate their DNA, so that each daughter cell can receive the full genetic make-up from the mother cell. Only upon duplication, cell division occurs. DNA duplication is a carefully choreographed process, which is carried out by the "workers of the cell", large protein machineries, co-operatively assembled along many checkpoints to ensure faithful copying. Since the molecule DNA itself is shaped as a double helix, composed of two wound-up strands, it has to be unwound to grant access to the genetic information for the copy machinery. Unwinding is carried out by a tightly integrated complex of proteins, forming the enzyme called DNA helicase, but its activity can be occasionally misregulated, causing cellular stress or ageing, resulting in human disease or growth defects in plants. In the normal cell, the ring-shaped, pre-mature helicase is first being wrapped around DNA before its activation. We have recently uncovered detailed insights into the interactions of the pre-mature helicase with the protein that helps loading it around DNA and identified a stretch in a protein called Cdt1 that might act as a clamp to open and close the ring. The opening process is particular important later in the unwinding process and therefore has to be explored to extend our understanding of the normal versus an abnormal process. To study the helicase during the process of loading around DNA in vitro, we created several helicase variants that "freeze" the helicase loading process at specific points and enable us to study the outcome in detail using high-resolution cryo-electron microscopy and sophisticated computational methods. The mechanistic insight into accurate helicase loading onto DNA gained from the presented study will generate an overview of several essential early steps towards eventual unwinding of the DNA and DNA duplication. While this is particularly important for a basic understanding of how our cell works, it has important implications for human diseases such as cancer but also will more immediately support research into inhibitors of infectious diseases.

Impact Summary

Our work will have a large impact on a broad range of researchers in the academic sector, but also clinicians and engineers. A stepwise explanation of how the helicase interacts with its loading helpers would enrich the scientific community in academia and industry immensely. As with all research, a particular outcome is difficult to predict, but the research environment is set up fully to tackle this particular project. Our structural work on the highly disease-relevant helicase loading will inherently benefit the strong pharmaceutical and healthcare sector in the United Kingdom. Since basic research into cellular biology is rarely covered by these companies, our work is the essential foundation on which translational research is built upon. The subject of our work, the DNA helicase is of interest in relation to cancer, ageing, and particular syndromes or even as target for anti-fungal inhibitors. However, it also becomes clear from plant research, that helicase loading is critical for seed development, stress tolerance or cellular differentiation. Thus, our work could benefit the UK green industry. The researchers (postdoc, technician, students) carrying out the project will gain invaluable experience in state-of-the art cryo-EM sample preparation and modelling of structural data. In turn, training of the researchers will benefit the UK biotechnology and pharmaceutical companies as well as academic institutions at national and international level. To increase scientific and health literacy, we will increase our efforts to communicate our findings and methodologies to the general public, for example as part of the Wohl Reach Out Lab at Imperial.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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