Characterisation of the HELLS and Irc5 subfamily of chromatin remodellers

Abstract

ATP-dependent chromatin remodelling enzymes regulate the structure of chromatin and are important in the prevention of genomic instability and tumourigenesis. Inaccurately repaired or unrepaired double-strand breaks can lead to mutations, cell death and cancer. The Lsh subfamily of chromatin remodellers has been implicated in DNA repair, and mutations and overexpression of the human protein HELLS have both been linked with several types of cancer. Previous work from this group has shown HELLS is involved in the homologous recombination repair pathway particularly within heterochromatic regions of the genome. Biochemical activity of this subfamily is poorly characterised and further research into the properties of the mammalian HELLS and its budding yeast homolog Irc5 would aid the understanding of their role in vivo and permit assessment of their potential as therapeutic targets.
This thesis investigates the role of Irc5 in genome stability in yeast and finds that Irc5 genetically interacts with HR genes and that loss of Irc5 activity results in defects in some DNA repair pathways. Catalytic activity of Irc5 will be examined in vivo to further understanding of the importance of its activity. Further to this, this thesis outlines the purification of recombinant HELLS and Irc5 proteins, which permits analysis of these proteins in vitro. Recombinant HELLS and Irc5 display DNA binding activity with a preference for structured DNA and this work finds that HELLS and Irc5 on their own do not display nucleosome sliding activity. Finally, this thesis studies binding of HELLS to other proteins and identifies interactions with a DNA repair protein and components of the nucleosome that provide insights to its mechanism of function.
The work outlined in this thesis on both HELLS and Irc5 are consistent with the hypothesis that this subfamily plays a role in repair and genomic stability.

Date of Award	23 Mar 2021
Original language	English
Awarding Institution	The University of Bristol
Supervisor	Mark S Dillingham (Supervisor) & Anna L Chambers (Supervisor)