A molecular understanding of how MCM2-7 becomes loaded onto DNA to maintain genomic stability

Our cells contains DNA, representing a "genetic blueprint of life". DNA is composed of two complementary strands, which contain the genetic information in their centre. It is imperative that prior to cell division the DNA becomes duplicated, so that each daughter cell receives a full genetic complement from the mother cell. DNA duplication is a careful choreographed process, where numerous proteins synergize to assemble large DNA replication machinery at replication origins. Central for DNA duplication is an enzyme - the DNA helicase - that separates the two strands of DNA, to give copying machine access to the information that lies in the centre of the DNA. Initially the helicase must be loaded onto DNA, before it can become active and participate in DNA duplication. However, the helicase loading reaction is frequently misregulated during aging, cellular stress or in cancer, resulting in human disease or reduced crop yields in plants.
Fortunately, cells have evolved a highly efficient mechanisms that allows the loading of the helicase onto DNA. While this process in simple bacteria is well understood, our recent work has revealed that in eukaryotes this process is quite different. We have now uncovered evidence that the helicase itself is crucial for the loading process, which has not been seen to such an extent in bacteria. Now we want to uncover exactly how the helicase assembles on DNA and how it is loaded onto DNA, producing a ring shaped complex that encircles DNA.
We have created a number of helicase variants that "freeze" the helicase loading reaction at specific points. Additionally, we have developed several unique tools to analyse the detailed structural and functional changes associated with helicase loading. Therefore we propose for this programme to use these helicase variants to uncover how this molecular machine is able to be loaded in an accurate and processive way onto DNA and to define the organismal consequences of introducing the helicase variants in yeast cells.
Our work will generate a mechanistic framework, showing how helicase loading actually works. This will support the work of researchers in related fields, but could also lead to the development of helicase loading inhibitors with anti-fungal activity.

The precise duplication of chromosomal DNA is essential for preserving the genetic complement of the cell. During the first step of DNA replication, termed pre-replication complex (pre-RC) formation, the replicative MCM2-7 helicase is loaded onto double-stranded DNA. Efficient and regulated loading of the replicative helicase is vital to maintain genomic stability and to prevent premature aging, growth defects in plants and tumorigenesis. Inhibition of helicase loading can occur due to mutations in helicase loading factors, inappropriate DNA secondary structures or due to altered expression of inhibitory factors, while excessive amounts of helicase loading proteins are associated with DNA re-replication, inappropriate homologous recombination and general genomic instability. Although we have identified the essential factors for helicase loading the overall process is only poorly understood, and the crucial mechanisms underlying efficient helicase loading and complex assembly remain to be established.
The aim of the proposal is to address how the replicative is loaded in a processive manner around DNA. We will be using an array of biochemical, electron microscopy and in vivo approaches to uncover the underlying mechanisms of helicase loading, a central process in DNA replication. The use of novel helicase mutants that promote specific arrests during the multi-step helicase loading process, will be used in combination with sophisticated biochemical assays and structural approaches to uncover crucial aspects of the helicase loading process. Together these studies will generate a mechanistic framework, which will allow us to better understand how altered helicase loading results in disease, aging or reduced crop yields. Moreover, we will test if it is possible to inhibit helicase loading, which could lead to the future development of inhibitors with anti-fungal activity that may improve human health and well-being in the long-term.

The academic sector will benefit largely from the proposed project, through knowledge gain, development of new methods and technologies. The structural characterisation of large protein complexes by the PDRA in collaboration with Xiaodong Zhang (ICL) and Huilin Li (Brookhaven National Laboratory, NY, USA) will transfer some of this knowledge and technology to the group of CS and other associated groups - leading to enhanced research capacities. Application of this cutting edge technology will have positive impact on the DNA replication field and associated research areas, as it will allow the formulation of new research questions and will generate new results. The project will offer opportunity for career development and training, which will be readily transferrable to other related fields across the spectrum of Molecular Biology and Biochemistry. Indeed previous group members have been readily employed at research institutes, universities and in industry.
This project could have both short- and long-term socio-economic impacts for the United Kingdom. As with every research project the impact depends on the results obtained and is therefore difficult to predict. In the medium this project may spur the development of helicase loading inhibitors in yeast, thereby addressing a potential way to treat fungal infections. This could spur the commercialisation of scientific knowledge and products in the long-term. Related drugs that inhibit the loading of the human replicative helicase could generate new anti-cancer agents, which would benefit the UK. Importantly, the project would lead to employment of a PDRA, thereby contributing to the national economy. The interdisciplinary nature of the project will greatly enhance the training of the PDRA in high-end modern techniques ranging from biochemical, genetic to electron-microscopy based methods. Statistical analysis and modelling of structural and biochemical data will feature in the proposed research and the integration of the data from various data sets will demonstrate the power of this interdisciplinary approach. Such a trained PDRA (and associated PhD, technician, masters and undergraduate students) are likely to benefit UK biotechnology and pharmaceutical companies, as well as the university sector at national and international level. Finally, the general public will benefit from this work by becoming informed about our science in general and the project specifically. Indeed, we have planned a number of methods to engage with the public in different ways and to make our work as accessible as possible.