Properties, structure and dynamics of the telomeric nucleosome
Date of Issue2019-06-12
School of Biological Sciences
Telomeres, the ends of human chromosomes are known to be bound by the protective shelterin complex and form nucleosomes and chromatin but the details of telomeric chromatin at the molecular and atomic level are unknown. In vivo studies on telomeric chromatin show a uniform micrococcal nuclease pattern with a nucleosome repeat length that is 40 bp shorter than bulk chromatin (~157 bp). These differences from bulk chromatin along with the fact that telomeres are hotspots for DNA damage, begs the question of whether telomeres have a different chromatin structure to bulk chromatin. In vitro studies on telomeric nucleosome core particles (NCP) are limited. This thesis investigates the reconstitution, biophysical properties and structure of the telomeric NCP. Through this work, we hope to address the gap in our understanding of telomeric chromatin. The repetitive nature of telomeric DNA and its propensity to form G-quadruplex under physiological salt condition pose a challenge in preparation of telomeric DNA template of defined length for reconstitution. A modified DNA extraction protocol employing Mg2+ in place of Na+ and ion exchange purification yielded milligram amounts of pure telomeric DNA of defined length. We have successfully reconstituted a telomeric NCP with human histone octamer and 145 bp DNA comprising 23 repeats of TTAGGG sequences. Biophysical characterization using dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) shows that the telomeric NCP has a larger radius of gyration and increased DNA breathing comparing to ‘601’ high-affinity DNA sequence. Our comparison showed that among different NCPs tested, the telomeric NCP shows the lowest affinity for histone octamer and the lowest stability in salt-dependent dissociation assays. The crystal structure of telomeric NCP with 2.2 Å resolution was solved by molecular replacement followed by the rebuilding of DNA employing anomalous maps. DNA stretching on the telomeric NCP was different from that of the “601” (high-affinity nucleosome positioning sequence) and alpha satellite NCPs of the same length. Among the five dinucleotide steps in telomeric NCP, GG and GT steps which have the highest stacking energies occupy 9 of 12 pressure points at the minor groove facing histone octamer. Here we outline the biophysical properties and present the high-resolution structure of the NCP formed by telomeric DNA, which is thought to be a sequence with the least affinity for histone octamer. We hope the findings presented here add to our understanding of DNA packaging.