Determining the macromolecular structure and cellular function of an alternative MCM complex

In all forms of life on the planet, DNA, the genetic material, carries information from one generation to the next. Each time a cell divides into two, it must first duplicate its genetic material by a process known as DNA replication. For DNA replication to begin, the two DNA strands that make up the familiar coil of the DNA double helix must be separated. Central to this process of DNA unwinding is a "molecular machine" called the MCM complex. The MCM complex is indispensable for DNA unwinding during DNA replication and is composed of six individual protein sub-components called Mcm2, Mcm3, Mcm4, Mcm5, Mcm6 and Mcm7. The importance of MCM function for health is highlighted by the fact that impaired or perturbed MCM function can lead to the developmental defects and tumour formation. Indeed, MCM protein levels are already an important clinical marker for certain types of cancer. Understanding precisely how the MCM complex performs its function is a major focus of research worldwide.
The work proposed in this application focuses not on the well-known MCM complex, but on a little-studied alternative form of the complex, one in which one of the six MCM sub-components (Mcm2) is substituted by a different protein altogether (MCM-BP), and aims to address several key questions, specifically, what is the actual structure of this new, variant MCM complex, precisely what function does it perform in the cell, and crucially, how does it contribute to the maintenance of chromosome integrity and disease avoidance? To address these questions, a simple model system will be used: a single-celled yeast that is easy to grow in the lab and which is ideally suited to the type of experimental work that will be used to determine the structure and cellular function of the new complex. The results obtained from analysis of the yeast complex will shed considerable light on the function of the corresponding complex in human cells and provide insights into the likely impact on human health when the function of the complex is impaired. In the longer term, the results of this study could lead to improved diagnosis and treatment of genetic diseases including cancer.

Defective DNA replication can have a wide variety of effects on genome structure and information content, such as sequence deletion, insertion and duplication, point mutation and chromosome fusion. In mammals, defects in chromosome replication can lead to developmental defects, growth impairment and tumour formation. The MCM complex plays an essential role in eukaryotic chromosome replication as the catalytic core of the CMG complex, the enzyme that unwinds double-stranded DNA at the replication fork. A number of studies have drawn direct links between MCM dysfunction, chromosome instability and disease, including cancer.
Recent evidence points to the existence of an alternative form of the MCM complex, in which one subunit (Mcm2) is replaced by the MCM-BP protein. The work proposed in this application aims to determine the structure of this alternative MCM complex, to investigate its function, and to establish how this function impacts on that of the archetypal MCM complex. Using fission yeast as model, the MCM-BP complex will be purified and its size and subunit composition determined by biochemical and biophysical methods. EM will be used to visualise 3D structure, and state-of-the-art protein crosslinking techniques used to map subunit-subunit interactions. The ATPase and DNA helicase activities of the complex will be examined using a variety of artificial fork-like substrates. To complement these biochemical approaches, the cellular function of the complex will be probed using a variety of molecular and cell biological methods. DNA combing will be used to measure replication origin firing, fork migration and recovery from fork stalling in MCM-BP mutant cells at the single molecule level, effects on chromosome cohesion will be monitored and the efficiency of fork recovery after stalling assayed. Overall, this work will lead to an understanding of the role played by the MCM-BP complex in the maintenance of genome stability and disease avoidance.

Obtaining a detailed understanding of the molecular mechanisms that underlie genome integrity is crucial for understanding how mutations in genome stability factors cause disease and can make a vital contribution to the development of methods for accurate diagnosis as well as to the development of effective disease treatments. The proposed work focuses on the conserved MCM-BP complex that plays an important, but poorly understood, role in the maintenance of genome integrity in eukaryotes. The MCM-BP complex is related to the well-studied MCM complex, mutation or perturbation of which can lead to developmental defects and cancer. Probing the function of MCM-BP complex using a simple model organism, and understanding how the function relates to that of the well-studied MCM complex, will lead to a better understanding of the role of the complex itself, its relationship with the well-studied MCM complex and its potential role in human disease. Thus, the main societal impact of the proposed research lies in its potential to contribute to the nation's health by spotlighting the role of the MCM-BP complex in the maintenance of genome integrity, inspiring research on the human MCM-BP complex, and in the longer term, potentially opening up new opportunities for disease management. Economic impacts of the proposed work lie in the opportunities for commercialisation and exploitation of the results of this and future studies in the development of diagnostic tools and therapeutics.