Viewed through a microscope, cells undergo a spectacular transformation as they enter mitosis, the phase of their existence just before they divide. The formation of the mitotic spindle, where the cell?s network of microtubule fibres is completely rearranged to span from either end to the chromosomes at the centre, is particularly striking. This spindle is a molecular machine that ensures the cell?s chromosomes are accurately distributed between its two daughter cells. Errors in the workings of the spindle are a known driving force of cancer and are also responsible for a congenital brain disease. Several control mechanisms ensure the mitotic spindle is normally assembled correctly. At an early stage in assembly, two proteins called TACC3 and ChTOG promote microtubule stability and hence promote assembly. These proteins are more effective when TACC3 is modified by a phosphate group: one phosphorous atom and three oxygen atoms that is commonly used by cells to alter the activity of their proteins. In the case of TACC3, the protein that adds the phosphate is called Aurora-A. Spindle assembly is thus controlled by the activity of Aurora-A, which is itself controlled by many other proteins under the influence of events within and outside the cell. We propose to investigate how the phosphate group influences the effectiveness of the TACC3/ChTOG partnership at the level of atoms. How this works is currently a mystery as the phosphate is only four atoms big, and yet it changes the activity of TACC3/ChTOG which total tens of thousands of atoms. We will use electron microscopy, a technique that allows us to see directly the shapes of proteins, to study the changes in TACC3 upon phosphorylation, and the effect on ChTOG. We will also use X-ray crystallography to determine the location of every atom within the proteins and to map the atoms by which TACC3 and ChTOG cooperate. This information will allow us to make a hypothesis for the details of how the TACC3/ChTOG partnership works and how phosphorylation enhances their effectiveness. We will use our protein structure models to design subtle modifications to TACC3 and ChTOG to test this hypothesis in human cells grown in culture. An overabundance of TACC3, ChTOG or Aurora-A have been linked with cancer, and TACC3 and Aurora-A are also important in brain development. These studies will provide the impetus for future investigations to understand the role of these proteins in human disease.

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Each human telomere is composed of 10-15 kb of repetitive DNA bound by a protein complex called shelterin, which forms a protective nucleoprotein cap at the chromosome end. In addition to shelterin, telomeric DNA is also wrapped around histone octamers in a closed, heterochromatic state that compacts telomeric DNA and represses transcription at the chromosome end (Tardat and Dejardin, 2018). Mutations that disrupt the assembly of chromatin at telomeres cause DNA damage and are found in essentially all 'ALT' type cancer cells (some 10-15 % of all tumour types), underlining the importance of chromatin in the function of telomeres. At non-telomeric sites, chromatin is assembled during S-phase when chromatin remodelling factors and histone chaperones disassemble nucleosomes in front of the replication fork and reassemble them on newly synthesised DNA (Hoek and Stillman, 2003). Although telomeric chromatin is also assembled during S-phase, genetic studies show that a distinct set of chromatin remodelling factors are required for this process, suggesting the replication and reassembly of nucleosomes at telomeres occurs through a distinct mechanism. The successful candidate will examine this mechanism using a combination of reconstitution biochemistry, biophysics and genetics. The starting point for the project is a reconstituted system for DNA replication that we have recently developed in the Telomere Biology lab. Combining this system with chromatinised DNA templates and purified chromatin remodelling factors, we will examine i) how chromatin affects the human replication fork ii) how telomere-specific chromatin remodelling factors allow replication and reassembly of nucleosomes on telomeric DNA and iii) the consequences of disrupting these processes within cells. As opportunities arise, we will also collaborate with other groups to characterise replication intermediates using cutting edge biophysical and structural techniq

Most inhibitor-bound protein structures used in structure-based drug design are determined using crystallographic data from single crystals collected at cryogenic temperatures. However, it remains difficult to routinely obtain single protein crystals large enough for high resolution data collection. This project aims to investigate if multicrystal, room temperature X-ray crystallography, in which X-ray data from many small crystals is collected at room temperature and merged to a complete dataset, is suitable for application in structure-based drug design. Differences in compound binding and differences in X-ray induced radiation damage to bound ligands between data collected at cryogenic- and room-temperature will be investigated.

See Section 9 of the report