Biochemical analysis of activation-induced cytidine deaminase (AID) from bony fish: insights into the mechanisms of AID

Dancyger, Alex (2012) Biochemical analysis of activation-induced cytidine deaminase (AID) from bony fish: insights into the mechanisms of AID. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (25MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

The properties of Activation-induced cytidine deaminase (AID) necessary for its role in immunity and lymphoma generation are currently unknown. AID mutants and species-variants, as compared to wild-type human AID, were identified so as to map out responsible residues/domains. Variant AIDs were analyzed by alkaline cleavage and EMSA. AID from bony fish species (Ictalurus punctatus and Danio rerio) were identified as the most divergent from human AID. Dr-AID acted enzymatically similar to H-AID, while Ip-AID was less mutagenic. Point mutations within Ip AID were generated, substituting the human counterpart amino acid. One mutant rescued Ip-AID enzyme activity to levels seen in H-AID and Dr-AID. The reverse mutation in H-AID almost fully abolished the deaminating ability of H-AID. DNA-binding kinetics were tested at different temperatures to analyze if the disparity in activity levels was due to binding of DNA. The single mutation in IP-AID increases the affinity of AID to bind ssDNA and this effect is substantially more pronounced at lower temperatures. These results have provided novel information on AID domains/residues necessary for its function.

Actions (login required)

View Item