Investigating the interaction between DNA-PK, cGAS and IFI16 in the innate immune response pathway

DNA-dependent protein kinase (DNA-PK) is a complex of proteins that acts as a crucial component of the non-homologous end joining (NHEJ) mechanism of DNA double-strand break repair. However, recent studies suggest that DNA-PK may have an additional secondary role in regulating the innate immune response to viral infections. These studies indicate that DNA-PK may regulate the type 1 interferon response by interacting with cGAS and through the cGAS interacting with IFI16, leading to the STING pathway. The experimental evidence of interactions between cGAS and DNA-PK as well as between IFI16 and cGAS suggests that both IFI16 and DNA-PK may be important regulators of the type 1 interferon response. This PhD project will aim to elucidate whether DNA-PK and/or IFI16 regulate the cGAS-STING pathway, by examining whether there are direct interactions between these proteins. This will involve protein expression, purification and biochemical/biophysical techniques to determine protein and DNA interactions. Through this experimental work, this project will help determine whether DNA-PK or IFI16 bind to cGAS and, if both bind, whether these proteins bind to cGAS simultaneously to form a single complex. Ultimately, cryo-electron microscopy will be utilised to study the structure of these complexes and determine their specific interactions. This work will help establish whether these proteins regulate the innate immune response to viruses and will provide an indication of the mechanism through which this occurs. Consequently, this project will determine the effect that DNA-PK and IFI16 have on the intensity/efficacy of viral clearance within infected host cells. Furthermore, understanding these interactions and the mechanism will help guide development of future vaccines and antivirals to enable new strategies for combating viral infections.