Design and Application of a Mutational Sensor for Characterizing AID Activity

Abstract

Activation‐induced cytidine deaminase (AID) is a mutator enzyme that introduces uracil:guanine mismatches in Immunoglobulin (Igh) genes during the antibody diversification processes of class switch recombination and somatic hypermutation in B lymphocytes. When this happens, one of three pathways is possible. Firstly, the DNA can be directly replicated with the mutation, causing a transition (C → T) mutation. Secondly, during genomic mutation, the repair enzyme Uracil N‐Glycosylase (UNG) can act to base excise the mutation in either an error free fashion, replacing the original cytidine in the mutated spot, or thirdly, an error prone fashion, in which a transversion mutation (C → G/A) will then be introduced. Certain “hotspots” in the DNA sequence increase mutational frequency: cytosine residues are strongly favored when they are part of an RGYW sequence, or its reverse complement, sequence WRCY (where W=A/T, R=A/G, and Y=C/T). However, AID can also produce off target damage to genes, including oncogenes, that result in lymphomas. Therefore, tight regulation of AID mutator activity is necessary for genomic stability. In this study, we designed a cellular mutation reporter assay to determine the role of AID in regulating mutational activity. Mutations introduced by AID were measured with the use of constructs containing a green fluorescent protein (GFP) with premature STOP codons which‐ upon mutation‐ will “turn on” GFP and can be screened by flow cytometry. The use of this construct allows for mutational screening to be done in any cell type in which AID is naturally occurring, such as lymphomas and leukemias, rather than having one cell type with the reporter gene built into the cell line (current method). Once the constructs were successfully created via PCR SOEing reactions, we verified their ability to serve as mutational sensors. To achieve this, cells were co‐transfected with constructs and AID into BOSC cells and analyzed for rates of nucleotide reversion via flow cytometry. AID is shown to reproducibly revert two constructs with premature stop codons in contextual hotspots from GFP− to GFP+, as seen by the increased presence of GFP when analyzed via flow cytometry. The data show that the GFP mutational sensor works and serves as a functional system for determining mutational analysis among BOSC cells via flow cytometry analysis.Support or Funding Information1. Allison Woods is an undergraduate scholar supported in part by the MARC Program at St. Mary's University and the Summer Program in Cancer Research at MD Anderson Cancer Center support by National Cancer Institute (R25CA181004).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.