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

The rotation will be within the Bolt research group, collaborating closely with the Allers and Gray groups. The Bolt group use bacterial and human cells to investigate DNA repair mechanism that is coupled to DNA replication. The Allers group investigate similar systems in archaea, and the Gray group in yeasts.
The overall aim of the project is to manipulate the structure of a small protein (RusA) and utilize it for physical detection of DNA repair structures called Holliday junctions. Their formation can be detected from human, bacterial, archaeal and yeast cells but there is a need to visualise them directly in real-time to understand DNA repair dynamics. RusA is an outstanding candidate for use in all four of these cell types. The rotation will comprise:
Design and generation of DNA constructs for fluorophore-RusA expression in human- U2OS; archaea - Haloferax volcanii; bacteria - Escherichia coli; yeast - Saccharomyces cerevisiae
Training in techniques of interpreting and manipulating protein structure
Training to over-express proteins in all four cell types, in preparation for fluorophore-RusA
Full Project Description:
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