Investigation into the structure dynamics and remodelling of the nucleosome using site-directed spin labelling and EPR distance measurement.

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The genomes of eukaryotes are packaged within the confines of the nucleus as a condensed structure termed chromatin. The nucleosome is the fundamental repeating subunit of chromatin. It consists of an octamer of four, core histone, proteins around which 146 bp of DNA is wrapped in nearly two turns. As a consequence all genetic processes in eukaryotes must contend with nucleosomes. For example, there are cellular mechanisms dedicated to modulating the dynamic properties of nucleosomes during the transcription cycle. These act to both improve and restrict access to the underlying genetic information as, and when, required. It is likely that the physical properties of the nucleosome are finely tuned to meet the apparently conflicting requirements of reducing inappropriate gene expression while allowing permitting transcription when required. Over the last ten years our understanding of the nucleosome has developed greatly due to the determination of X-ray crystal structures of the nucleosome. However, during assembly and remodelling, chromatin exists in different forms for which high resolution structures do not exist. Understanding of how chromatin structure is manipulated during the course of gene regulation is now limited by a lack of structural information regarding these intermediates. In order to address this a suitable technique would ideally be, carried out in aqueous solution (or frozen solution), be non-destructive (so we can add components in stages and look for change), provide accurate and suitable distances with limited interference to the underlying structure, and be able to give some indication of molecular dynamics. We propose to use a technique called Electron Paramagnetic Resonance (EPR) to study structure of a number of chromatin assemblies. Using established techniques we will introduce spin labels into specific places on either the histone proteins or DNA fragments. Spin labels provide signals in the EPR spectrum and by use of relatively new techniques we can measure the distance, between the spin labels, over distances of between approximately 2nm and 8nm. ( The diameter of the nucleosome is approximately 100A and its depth 50A) By triangulation these distance measurements allow us to build up a picture of the structure of the molecules containing the labels. In our experience of model systems, the distance measurements are likely to have an accuracy of around 0.1nm. This means that it will be possible to determine how changes in the composition of a complex like the nucleosome, lead to an overall changes in the structure of the complex. Such measurements will be made on the nucleosome and related structures in an attempt to fill vital details that are not described by the available crystal structures. Because the core of the nucleosome is made up of 4 protein dimers, the system is ideally suited to investigation by EPR but poses severe problems for a technique that could give similar measurements, called Fluorescence Resonance Energy Transfer (FRET). In EPR we can measure the distance between two identical spin labels. These labels are generally smaller, and less disruptive that the fluorescent labels used in FRET. As there are two copies of each histone protein in the nuclesome, it is technically very difficult to make nucleosomes labelled with single donor and acceptor dyes. In contrast, selection of a single labelling site on a dimeric histone that is separated by a suitable distance when assembled into a nucleosome, followed by spin labelling, provides a simple means of generating substrates labelled with two identical dyes that is suitable for EPR measurements. The most valuable information obtained by EPR is the measurement of distances between labelling sites. This is typically carried out in a frozen solution of between 10-200uM concentration. We have experience in using EPR to make distance measurements on DNA, Protein, DNA-protein and RNA-protein complexes.