Abstract
DNA deaminases underpin pathways in antibody diversification (AID) and anti-viral immunity (APOBEC3s). Here we show how a high-throughput bacterial papillation assay can be used to screen for AID mutants with increased catalytic activity. The upmutations focus on a small number of residues, some highlighting regions implicated in AID's substrate interaction. Many of the upmutations bring the sequence of AID closer to that of APOBEC3s. AID upmutants can yield increased antibody diversification, raising the possibility that modification of AID's specific activity might be used to regulate antibody diversification in vivo. However, upmutation of AID also led to an increased frequency of chromosomal translocations, suggesting that AID's specific activity may have been limited by the risk of genomic instability.
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Acknowledgements
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, to J.-M. Buerstedde (Munich) for providing the DT40 cell lines, to O. Perisic (Cambridge) for providing the pOPTG vector, to Silvestro G. Conticello (Istituto Toscano Tumori) for helpful suggestions and the James Baird and Frank Elmore funds for support to M.W.
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M.W. and Z.Y. performed the papillation assay and selection of human AID upmutants; M.W. performed all other experiments; C.R. designed and assisted with the class-switching assay and translocation assay; M.S.N. designed the overall research and wrote the manuscript.
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Wang, M., Yang, Z., Rada, C. et al. AID upmutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity. Nat Struct Mol Biol 16, 769–776 (2009). https://doi.org/10.1038/nsmb.1623
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DOI: https://doi.org/10.1038/nsmb.1623
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