dc.contributor.advisor | Gilbert, Nicholas | en |
dc.contributor.advisor | Wood, Andrew | en |
dc.contributor.author | Wheeldon, Hannah Amy | en |
dc.date.accessioned | 2020-03-11T14:22:12Z | |
dc.date.available | 2020-03-11T14:22:12Z | |
dc.date.issued | 2020-06-27 | |
dc.identifier.uri | https://hdl.handle.net/1842/36855 | |
dc.identifier.uri | http://dx.doi.org/10.7488/era/157 | |
dc.description.abstract | DNA in the eukaryotic cell is organised into a complex called chromatin, which protects the
DNA from damage and allows the careful regulation of the genome. Precisely how the
genome is used by the cell is dependent on the structure and regulation of this complex, and
discovering how this is organised is therefore one of the principle challenges of modern
biology.
DNA is wrapped around histone octamers to form arrays of nucleosomes, which are
subsequently folded into a higher-order structure, speculated to be a 30-nm fibre. In vitro
studies of higher-order chromatin fibre structure have provided valuable information about
this structure, but it is not well understood how changes to the DNA sequence might affect
the structure and dynamics of the complex. DNA sequence is known to affect nucleosome
binding strength and positioning within an array, and I therefore hypothesised that DNA
sequence changes are likely to impact higher-order chromatin fibre structure. Using an in
vitro model of chromatin fibre structure, reconstituting purified DNA with purified core
histones by salt dialysis, allowed me to isolate the effects of DNA sequence in the absence of
confounding factors such as transcription factor binding.
I compared the higher-order chromatin structure and dynamics of the well-studied “601”
DNA repeat sequence with two novel reconstitution templates which contain biologically-derived
nucleosome positioning sequences. Sucrose gradient sedimentation of folded
chromatin fibres suggested that non-601 fibres may be as compacted as 601 fibres, but have
more heterogeneous structures. However, non-601 fibres were more easily perturbed under
tension than 601 fibres, suggesting that such sequences might promote a more accessible
chromatin environment. While repetitive 601 fibres were found to have a regular
nucleosome repeat length by DFF digestion, non-repetitive, biologically-derived sequences
had a more heterogeneous nucleosome repeat length, which I suggest is responsible for their
increased accessibility.
I also found that the compacted higher-order structure of the 601 fibre is disrupted by the
introduction of a single sequence with low affinity for the histone octamer. These structures
can be separated by sucrose gradient sedimentation, and I suggest that this could be a useful
method to examine the individual effects of a wider range of DNA sequences on higher-order
chromatin fibre structure in vitro. | en |
dc.language.iso | en | |
dc.publisher | The University of Edinburgh | en |
dc.subject | folded chromatin fibre | en |
dc.subject | non-repetitive DNA sequences | en |
dc.subject | 601 fibres | en |
dc.subject | histone octamers | en |
dc.subject | sucrose gradient sedimentation | en |
dc.subject | higher-order chromatin fibre structure | en |
dc.title | Impact of DNA sequence on the structure and dynamics of the higher-order chromatin fibre in vitro | en |
dc.type | Thesis or Dissertation | en |
dc.type.qualificationlevel | Doctoral | en |
dc.type.qualificationname | PhD Doctor of Philosophy | en |
dc.rights.embargodate | 2021-06-27 | |
dcterms.accessRights | Restricted Access | en |