Analysis of activation induced cytidine deaminase using atomic force microscopy

Gale, David J.G. (2021) Analysis of activation induced cytidine deaminase using atomic force microscopy. Masters thesis, Memorial University of Newfoundland.

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Abstract

Activation induced cytidine deaminase (AID) plays a large part within the pathway of immunoresponse. It does so by inserting mutations, during DNA replication, in B cells (immune system cells). This results in a more diverse set of antibodies in each subsequent generation. However, mutations that cause infinite replication with no cellular apoptosis, can lead to cancer. AID has been implicated in cancers which appear independent of the expected environmental causes, such as smoking or UV exposure. The method for AID mutations involves binding to single-strand DNA. Using Atomic Force Microscopy (AFM) we obtained both the geometry or topology, as well as the nanomechanical information about this mutation process and AID. In this thesis, I present the first AFM images of AID, showing direct structural information about the protein. Although the measurements are done in vitro, physiological conditions have been approximated in order to get an accurate analysis of the protein system. To allow imaging in buffer, suitable substrates were tested and identified which would bind the protein sufficiently in this high ionic strength environment. Optimized plating and scan conditions for AID in both wet and dry conditions are described, which allow high resolution imaging to be performed that is not often seen in biological systems. Through the many scans and procedural changes it was established that although difficult it is possible to gain an image of AID through the use of AFM. Using the some of the forces with the protein, an image of HOPG anchored AID was able to be obtained

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