We are looking for an ambitious PhD student with interests in learning about structural biology and DNA repair. Our goal is to gain a greater understanding of DNA repair to develop future therapeutics for treatment of diseases, ageing and cancer. Our work focuses on DNA double-strand breaks (DSBs). This type of DNA damage can be catastrophic for cell survival as if not repaired correctly it can lead to deletions, translocation, cell death and cancer. Complex processes have evolved to repair this type of DNA damage, including a mechanism called non-homologous end joining (NHEJ). Targeting NHEJ in combination with radio- and chemotherapy has proved successful in treatment against cancer. However, the molecular details of this process are still not completely understood. Understanding the structural mechanism of NHEJ has been a long-standing challenge, however recent advances in cryo-electron microscopy have begun to allow us to visualise large complex multicomponent assemblies involved in this process (Chaplin et al., 2020, Chaplin et al., 2021, Chen et al.,2021).
Although the architecture of the core proteins has been recently described, many questions remain to fully understand this mechanism. These include: i) what is the role of nucleases and polymerases. ii) what is the role of accessory proteins and how do they regulate or compete for similar sites. iii) How may these proteins arrange in vivo in relation to the physiological packing of DNA within nucleosomes and/or chromatin. iv) How does the structure and function of human NHEJ proteins compare to plants, archaea, and bacteria.
To answer these questions the project will provide training in many biophysical techniques such as ligation and kinase assays as well as electromobility shift assays, isothermal titration calorimetry and analytical gel filtration etc. These methods will first establish protein-protein and protein-DNA interactions. Once confirmed multicomponent protein samples will be prepared for cryo-EM analysis. This will provide hands-on experience with sample preparation, screening, data collection and processing in this exciting technique that has recently undergone a revolution, allowing for ever more detailed structures. Moreover, interactions with nucleosomes and chromatin will be evaluated to understanding the organisation in vivo. Finally, computational approaches will also be utilised to compare proteins involved in NHEJ across all kingdoms of life.
This is an exciting, highly rewarding, and competitive research area with direct implications for human health. Gaining further mechanistic and structural insights into NHEJ will aid future developments for therapeutics to combat diseases.
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