RNA enhances the activity of purified AID on DNA sequences of Immunoglobulin switch regions

Abdouni, Hala (2017) RNA enhances the activity of purified AID on DNA sequences of Immunoglobulin switch regions. Masters thesis, Memorial University of Newfoundland.

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Abstract

Activated B lymphocytes in peripheral lymphoid organs undergo class switch recombination and somatic hypermutation of antibody genes to produce antibodies with higher antigen-binding affinity. These processes are initiated by the DNA mutator enzyme activation–induced cytidine deaminase (AID) which mutates cytidine to uridine at Immunoglobulin loci. AID activity also results in mutations across the genome which in turn causes and exacerbates cancers. In vitro, AID mutates single stranded but not double-stranded oligonucleotide DNA substrates whereas in vivo, it acts preferentially on loci that are robustly transcribed or contain unusual transcriptional features. Taken together, AID is thought to target single stranded DNA liberated during transcription. In addition, AID has been shown to interact with factors associated with transcription, including the RNA exosome and spliceosome components, and with non-coding RNA itself. Here, we examined the behavior of purified AID on DNA/RNA hybrid substrates designed to simulate structures found at Immunoglobulin loci. We found that when composed of Immunoglobulin switch sequences, DNA/RNA hybrids significantly boost the catalytic activity of AID. We also identified numerous AID mutants which are catalytically inactive on DNA, whose activity is restored by proximity to RNA. Given the prevalence of RNA:DNA hybrids at Immunoglobulin switch sequences, our finding that RNA directly enhances the catalytic activity of AID on DNA suggest a novel role for RNA in regulating AID activity.

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