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Title: DNA-PKcs/Artemis complex in DNA double-strand-break repair : cryo-EM and biochemical studies
Author: Liang, Shikang
ISNI:       0000 0004 8508 4441
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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DNA is the main carrier of inheritance and DNA damage will thus have serious consequences. DNA double-strand breaks (DSB) are amongst the most lethal forms of DNA damage. One DSB can lead to catastrophic consequences including cancer and cell death. To fix it, there are two main mechanisms-- homologous recombination (HR) and non-homologous end joining (NHEJ). My PhD project focuses on NHEJ. Unlike HR, NHEJ is not limited by the cell cycle as it does not require a template for recombination. Also, NHEJ has been shown to be the preferred DSB repair pathway in higher eukaryotic organisms including human. NHEJ is dynamic and flexible but can be separated into three steps - DNA end recognition, end synapsis and processing, and end ligation. The main objective of my project is to understand the interaction in the complex between DNA-PKcs and Artemis, which is the major nuclease in the step of end synapsis and processing to help clean up the modified DSB ends caused by external factors including ionizing radiation. It is also the only discovered human endonuclease cleaving hairpin DNA, which is indispensable in V(D)J recombination— a mechanism that provides the immunodiversity of antibodies and T-cell receptors. During my PhD, I purified Artemis and DNA-PKcs, conduct biochemical and biophysical characterisation of these proteins and used cryo-electron microscopy (cryo-EM) as the main structural method. The nuclease assays for investigation of endonuclease activity targeting hairpin DNA revealed that XLF and XLF/XRCC4 have a stimulating effect on the endonuclease complex without and with Ku. Moreover, I have identified the region of Artemis interacting with DNA-PKcs and collected cryo-EM data for complexes of DNA-PKcs with different Artemis constructs, revealing the interaction mode between DNA-PKcs and Artemis. In addition, I further explored the cryo-EM structure of the DNA-PKcs to provide a firmer ground for modelling and the related complex study. A part of my PhD project focuses on the preliminary drug discovery of Artemis/ DNA Ligase IV complex. An intrinsically disordered Artemis C-terminal peptide interacts with DNA Ligase IV through concerted folding, showing a site that can be most easily targeted by small molecules. Therefore, during my PhD, different constructs of DNA Ligase IV have been screened and the fragment-based drug discovery approach was initiated to provide chemical tools or candidate drug molecules to inhibit the Artemis-DNA Ligase IV binding site. Moreover, collaboration work with the Strick group enabled us to monitor the temporal organisation of NHEJ using the single-molecule method, which identified the function of PAXX as an early participating component in end synapsis.
Supervisor: Blundell, Tom Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: DNA repair ; DNA double-strand break ; Non-Homologous End Joining ; Cryo-EM ; DNA-PKcs/ Artemis complex ; NHEJ spatial organisation ; NHEJ temporal organisation ; Structural Biology ; structure-based drug discovery