Eukaryotic genomes are typically covered by a landscape of bound nucleosomes to form higher-order chromatin. Access to the DNA requires the remodelling of these nucleoprotein structures, resulting in histone ejection or nucleosome displacement. Many DNA helicase-like ATPases play a role in these processes. Rather than separating DNA strands (i.e., classical helicase activity), the remodelling ATPases are thought to interact with intact dsDNA and use ATP hydrolysis to produce mechanical force which is exerted on the nucleosomes. Although remodelling is key to genome replication, gene expression and DNA repair, we currently have an incomplete understanding of the molecular mechanisms of these motor enzymes. In collaboration with Anna Chamber at the University of Bristol, this project aims to provide a more detailed view of how ATP hydrolysis is coupled to nucleoprotein remodelling by the Irc5/HELLS sub-family.
The mammalian protein HELLS (also known as SMARC6 and LSH) is essential for maintaining DNA methylation markers and for efficient repair of double-strand breaks in DNA. It is mutated or mis-regulated in several human cancers. The budding yeast homologue Irc5 is also believed to play a role in regulation of DNA repair. However, remodelling activity is yet to be demonstrated for either protein. The aim of this project is to characterise how and why the ATPase activity of the Irc5/HELLS sub-family is coupled to DNA and nucleoprotein substrates. You will principally use yeast Irc5, purifying the protein and exploring its activity using in vitro enzymatic approaches. These will involve assays of nucleosome positioning, ATPase activity and movement on DNA. A key aim will be the development of single molecule microscopy assays to directly observe how the substrate is modulated during the ATPase cycle. Damage-specific interaction partners can then be introduced into the assays to determine how they modulate lrc5 activity.
To complement the analysis of the eukaryotic proteins, the project will also utilise a simple bacterial model system (a single polypeptide ATP-dependent restriction enzyme) for which we have X-ray structural data. This will help in development of the assays and techniques necessary to study the movement of a helicase-based motor along intact DNA whilst remodelling a nucleoprotein structure. Keywords: chromatin, DNA repair, gene regulation, cancer, ageing, single molecules, enzyme, helicase