Willmann, K.; (2012) Investigation of transcriptional targeting and DNA lesion resolution of Activation Induced Deaminase by cofactor analysis. Doctoral thesis , UCL (University College London).

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Abstract

Activation Induced Deaminase (AID) is a crucial protein in B cells, required for a functional humoral immune system. The regulation and the molecular mechanism of AID is the focus of my thesis. As an enzyme, AID induces mutations and recombination in immunoglobulin genes, leading to increased antibody affinity for antigen and alterations in antibody-effector function. Catalytically, AID deaminates cytosine to uracil in single-stranded DNA, a DNA lesion that leads to a mismatch in double-stranded DNA. AID can be controlled via multiple pathways, including: a) targeting AID to the correct locus and b) regulating the processing of the AID-induced lesion. Proteins proposed to be involved in targeting AID include members of the transcription and chromatin regulation machinery, whereas a variety of DNA repair and DNA metabolism proteins have been shown to be involved in AID-induced lesion-processing. The physical and functional interactions of AID with these co-factors are not well characterised. In this thesis I determined the molecular details and biology of candidates for AID interaction. First, I used a novel technique to uncover and characterise interacting partners of endogenous chromatin-bound AID from B cells. The presented work shows that the RNA polymerase II elongation associated protein PAF1 binds directly to AID, while knocking down members of the PAF complex in B cells inhibits AID-induced immunoglobulin diversification. Secondly, I was able to identify a novel interaction of the DNA repair regulator PCNA with AID, both in vitro and in vivo. Importantly, PCNA function was proven to be susceptible to interference by AID and thus influencing lesion resolution. Taken together, this thesis highlights the importance of cofactors for defining the AID molecular pathway, relevant to all known functions of AID within and possibly beyond the B cell reaction.

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