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AID upmutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity

Nature Structural & Molecular Biology volume 16, pages 769–776 (2009)Cite this article

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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Figure 1: Papillation screen for active mutators.
Figure 2: Dynasty of AID upmutants selected by papillation screen.
Figure 3: Nature of the AID upmutations.
Figure 4: Upmutants of fugu AID.
Figure 5: Enhanced antibody diversification by AID upmutants.
Figure 6: Increased chromosomal translocations by AID upmutants.
Figure 7: Comparison of AID with APOBEC3s.

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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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  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, UK

    Meng Wang, Zizhen Yang, Cristina Rada & Michael S Neuberger

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  1. Meng Wang
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  2. Zizhen Yang
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Contributions

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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Correspondence to Michael S Neuberger.

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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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