The investigation of nucleosome dynamics using site directed spin labelling and electron paramagnetic resonance. (360G-Wellcome-080736_Z_06_Z)

WT Studentship for Mr Andrew J Bowman: 4-year PhD studentship in Molecular and Cellular Biology When nuclesomes were first discovered, it was thought they were static bodies, rigid complexes whose function was to package the genome into cellular dimensions. Contrary to this, within the last 15 years or so, nucleosomes have been shown to be highly dynamic entities. Widom and colleagues have shown that DNA wrapped around the histone octamer undergoes spontaneous, transient unwrapping, allowing access to DNA binding proteins. Studies carried out by the Prunell and Victor labs have indicated structural changes in the histone core, particularly in the (H3-H4)2-tetramer. In agreement with this, studies carried out by Cleveland and colleagues on the H3 variant CENP-A showed that certain regions of the tetramer have greater dynamicity. During my rotation project I employed the technique of Site-Directed Spin Labelling (SDSL) and Electron Paramagnetic Resonance (EPR) to further investigate the structural dynamicity of the (H3-H4)2-tetramer. The tetramer is a likely nucleosome folding intermediate, first associating with DNA to form a tetrasome, followed by the binding of H2A-H2B to form the nucleosome proper. However, there is no structural information on the tetramer or tetrasome as a free entity. Another region of the nucleosome which is lacking structural information, though of great biological importance, are the histone tails. It is the general consensus that histone tails are devoid of secondary structure, protruding from the nucleosome into solution as a random coil. In the crystal structures of nucleosomes all but one of the histone tails do not reside in defined positions, the exception making inter-nucleosomal contracts, and thus are not resolved.