The Role of DNA Damage Signalling in Adaptive Immunity

Abstract

Adaptive immunity is essential for the survival of many different organisms. Immune system mechanisms differ between species, but they all have two things in common; adaptability and memory of the immune response. These depend on regulated and often irreversible alterations to the host genome. DNA damage signalling and repair is therefore closely linked with adaptive immunity. In this thesis, I will present the first part of two projects dealing with adaptive immunity of bacterial and mammalian organisms. I will explore the novel repair factor BRD8 and its potential role in both the DNA damage response pathway and in antibody diversification of mammals. BRD8 was implicated in CSR in a genome-wide short hairpin RNA screen. Preliminary data further implicates BRD8 in both DNA damage repair and antibody diversification. Murine BRD8 is highly expressed in immune cells, specifically immune cells undergoing antibody diversification. Both human and mouse BRD8 share homology with and interact with various known DNA damage repair proteins. Further studies will help reveal its exact role in both DNA damage repair and antibody diversification. I will also use the bacterial adaptive immune system, Clustered regularly-interspaced short palindromic repeats (CRISPR)-Cas9 for gene editing. Modifications to the system has great potential for therapeutic and experimental uses. A model system based on 293T/GFP-puro cells was developed for rapid characterization of various Cas9 fusion variants we designed. RNA-deaminase Adar1 has been implicated in somatic hypermutation (SHM) [1], which in antibody diversification increases the affinity of an antibody for a specific antigen through mutations of the variable domain. I therefore fused the deaminase domain of Adar1 to the nuclease-deficient dCas9 in an effort to mimic somatic hypermutation ex vivo. The effects of Cas9 was assayed on the DNA level using a celery juice extract we have also developed and established. Preliminary data did not reveal any Adar1 deaminase activity on the eGFP locus, but ongoing studies seem more promising. A functional Adar1-dCas9 can potentially be used for in vivo single-base substitutions for both therapeutic and research purposes.