A High-Throughput Assay for DNA Deaminases
Most members of the AID/APOBEC family of polynucleotide deaminases can catalyse the deamination of cytosine to uracil in DNA. They thereby function as active DNA mutators. Here, we describe how bacterial papillation assays can be adapted to monitor the mutator activity of AID/APOBEC proteins and show how such papillation assays can be used as a high-throughput screen to identify AID variants with increased specific activity. It should also be possible to use papillation assays for the identification of novel DNA deaminases.
Key wordsPapillation assay Activation-induced deaminase APOBEC DNA deaminase Cytosine deamination
We are indebted to J. Miller (Molecular Biology Institute and Department of Biology, University of California, Los Angeles) for kindly providing E. coli strain CC102 and recommendations regarding plating, and Gareth Williams and Salome Adam (Cambridge, UK) for helpful comments on the manuscript, and the James Baird and the Frank Elmore funds for support to M.W.
- 23.Sambrook, J. and Russell, D. (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Woodbury, NY. 66, 75.Google Scholar