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Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions

Nature Immunology volume 12, pages 70–76 (2011)Cite this article

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Abstract

Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA.

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Figure 1: Strategy for identifying uracil residues in DNA.
Figure 2: Uracil residues in the rearranged Igl allele from UNG-deficient DT40 cells.
Figure 3: Uracil residues in VH and Vκ regions from mouse germinal center B cells.
Figure 4: Uracil residues in the Sμ region after ex vivo stimulation of mouse splenic B cells with LPS and IL-4.
Figure 5: DNA replication decreases the uracil content.
Figure 6: Uracil residues are present on both DNA strands in Sμ after ex vivo stimulation.
Figure 7: Detection of uracil residues by ligation-mediated PCR.
Figure 8: Uracil residues preferentially replace cytosine residues.

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Acknowledgements

We thank J. Stivers (John Hopkins University) for UNG; S. Wilson (National Institute of Environmental Health Sciences, National Institutes of Health) for polymerase β-lyase; R. Kohli (University of Pennsylvania), J. Buerstedde and H. Arakawa (Max Planck Institute of Biochemistry) for reagents and advice; S. Fugmann and R. Sen for discussions; T. Wolf, C. Nguyen and R. Wersto for assistance in flow cytometry; and the Comparative Medicine Section for mouse maintenance. Supported by the National Institute on Aging and the National Institute of Child Health and Human Development of the National Institutes of Health (Intramural Research Program) and Howard Hughes Medical Institute (D.G.S.).

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Authors and Affiliations

  1. Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA

    Robert W Maul, Huseyin Saribasak, Stella A Martomo, Rhonda L McClure, William Yang, David M Wilson III & Patricia J Gearhart

  2. Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

    Alexandra Vaisman & Roger Woodgate

  3. Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA

    Hillary S Gramlich

  4. Department of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA

    David G Schatz

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Contributions

R.W.M., H.S., S.A.M. and P.J.G. designed the study; R.W.M., H.S., R.L.M., W.Y., A.V. and H.S.G. did experiments; D.G.S., R.W. and D.M.W. provided reagents and suggestions; and R.W.M. and P.J.G. wrote the manuscript.

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Correspondence to Patricia J Gearhart.

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Maul, R., Saribasak, H., Martomo, S. et al. Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions. Nat Immunol 12, 70–76 (2011). https://doi.org/10.1038/ni.1970

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