Being seen on Holliday - a universal marker for comparing DNA repair by homologous recombination in multiple forms of life

The project will develop a new approach to identifying DNA repair by homologous recombination (HR), and deploy it in bacteria, humans, archaea, and yeast. HR comprises multiple related sub-pathways that overcome breaks in replicating DNA, with crucial roles in cancer biology, meiosis, and genetic flux in prokaryotes.
HR pathways in all organisms share common initial events that detect DNA strand breaks, resect them, and invade into homologous unbroken DNA. DNA breaks and resection can be detected by various means showing HR is underway. HR can then diverge into multiple sub-pathways. Defining these is complex in part because of indirect methods for their detection. We aim to directly detect one major pathway outcome - long tract HR by DNA replication that forms specialised DNA structures called Holliday junctions. This will help to understand exactly what factors direct and control HR pathway choice.
We will utilize the RusA protein to visualise Holliday junctions. This small (14 kDa) protein recognizes Holliday junctions with high specificity and cuts them to restore duplex DNA. RusA was originally identified in bacteria and has since been engineered with a nuclear localisation signal (NLS) and fused to a green-fluorescent protein (GFP) 'tag' for use in S. pombe yeast. In this PhD project we will use the catalytically active bacterial RusA enzyme alongside a mutant version (RusAD70N) that binds to Holliday junctions but is unable to cut them, therefore allowing Holliday junctions to persist. This will provide the means to visualise Holliday junction formation in response to DNA breaks.
First, we will detectably produce GFP-RusA and GFP-RusAD70N proteins with and without an NLS (as appropriate for cell type) in E. coli, S. cerevisiae, human bone osteosarcoma cells (U2OS) and the archaeon Haloferax volcanii. The proteins will be compared for visualising Holliday junction formation, by confocal microscopy (Bolt lab), during HR induced by various genotoxic agents, predicting that GFP-RusA foci should be increased in response to genotoxic stress. Each of these cell types and HR-inducing treatments are frequently used in the Bolt-Allers-Gray labs.
Establishment of GFP-RusA/RusAD70N imaging will facilitate the next stage of the project, to define factors in each cell type that divert HR towards Holliday junction formation. We will use genetics to delete or activate genes in each cell type that encode our candidate proteins. This is achieved using CRISPR/Cas9 editing in the human U2OS cells and by recombineering in S. cerevisiae, bacteria, and archaea - techniques already in place in the Bolt-Allers-Gray labs. Any genes identified as interesting in this context will then be scrutinised by introducing precisely defined point mutations using Cas9-RT 'Prime' editing in human U2OS cells, and 'Retron editing' in E. coli. These latter methods are not yet tested in yeasts or archaea, but we expect that they may also form part of this PhD project.
These investigations aim to identify new molecular mechanism in regulation of HR sub-pathways, that can be developed in further work in vitro.