Woods, Debbie Jane (2024) Mechanistic investigation of the SWI/SNF chromatin remodeller. PhD thesis, Birkbeck, University of London.
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Abstract
Chromatin remodellers are large multi-subunit complexes able to restructure the nucleosome, altering the accessibility of DNA for processes such as transcription, repair, or replication. SWI/SNF is an evolutionarily conserved ATP-dependent chromatin remodeller that uses the energy of ATP hydrolysis to move, remodel and evict nucleosomes. Mutations in various subunits of human SWI/SNF, BAF, have been found in 20% of human tumours, highlighting the complex as one of the most commonly affected targets in cancer. The aim of this project was to investigate the chromatin remodelling mechanism of SWI/SNF complexes by producing a “stalled” reaction intermediate of nucleosome bound remodeller for biochemical, biophysical, and structural analysis. We have successfully designed and developed a nucleosome substrate with a novel, single, site-specific DNA-histone cross-link capable of preventing the remodelling of exit side DNA, as demonstrated using a restriction enzyme accessibility assay. This thesis will also discuss the efforts to co-express recombinant S. pombe SWI/SNF in E. coli to provide a platform for structure-guided molecular genetics experiments. To follow the remodelling reaction at higher spatial and temporal resolution, a single molecule FRET assay has been established. These methods can be used to understand the effect of the nucleosome cross-link in remodelling and to inform future cryo-EM structural studies that aim to visualise transient intermediate states of BAF remodelling. The development of a “stalled" intermediate has broader applications to other fields of nucleosome study including transcription regulation and DNA replication, recombination, and repair.
Metadata
| Item Type: | Thesis |
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| Copyright Holders: | The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted. |
| Depositing User: | Acquisitions And Metadata |
| Date Deposited: | 09 Jul 2024 15:49 |
| Last Modified: | 09 Jul 2024 15:55 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/53798 |
| DOI: | https://doi.org/10.18743/PUB.00053798 |
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