Genetic and in vitro assays of DNA deamination

The DNA deaminase family encompasses enzymes that have been highly conserved throughout vertebrate evolution and which display wide-ranging positive effects upon innate and adaptive immune system and development. Activation-induced cytidine deaminase was identified as a DNA mutator after its necessity in the successful development of high-affinity B cells via somatic hypermutation, class switch recombination, and gene conversion was determined. APOBEC3 exhibits the ability to deaminate retroviral first strand cDNA in a variety of viral infections, including HIV and hepatitis. Recent work has highlighted the potential importance of activation-induced cytidine deaminase (AID) and APOBEC1 in epigenetic reprogramming, and also the role that AID and the APOBECs may have in the development of cancer. In addition to the known activities of these members of the protein family, there are still other deaminases, such as APOBEC2, whose targets and functions are as yet unknown. This chapter provides the details of two assays that have proved to be invaluable in elucidating the exact specificities of deaminases both in vitro and in Escherichia coli. The application of these assays to future studies of the deaminase family will provide an indispensible tool in determining the potentially diverse functions of the remainder of this family of enzymes.