The Journal of Experimental Medicine
ThymUS '08
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Published online December 10, 2007
doi:10.1084/jem.20071768
The Journal of Experimental Medicine, Vol. 204, No. 13, 3209-3219
The Rockefeller University Press, 0022-1007 $30.00
© 2007 Di Noia et al.
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ARTICLE

 Dependence of antibody gene diversification on uracil excision

Javier M. Di Noia1, Gareth T. Williams1, Denice T.Y. Chan3, Jean-Marie Buerstedde2, Geoff S. Baldwin3, and Michael S. Neuberger1

1 Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 QH, UK
2 Institute of Molecular Radiobiology, GSF -National Research Center for Environment and Health, D-85764 Neuherberg-Munich, Germany
3 Division of Molecular Biosciences, Imperial College, London SW7 2AZ, UK

CORRESPONDENCE Javier Di Noia: Javier.Di.Noia{at}ircm.qc.ca OR Michael Neuberger: msn{at}mrc-lmb.cam.ac.uk

Activation-induced deaminase (AID) catalyses deamination of deoxycytidine to deoxyuridine within immunoglobulin loci, triggering pathways of antibody diversification that are largely dependent on uracil-DNA glycosylase (uracil-N-glycolase [UNG]). Surprisingly efficient class switch recombination is restored to ung–/– B cells through retroviral delivery of active-site mutants of UNG, stimulating discussion about the need for UNG's uracil-excision activity. In this study, however, we find that even with the overexpression achieved through retroviral delivery, switching is only mediated by UNG mutants that retain detectable excision activity, with this switching being especially dependent on MSH2. In contrast to their potentiation of switching, low-activity UNGs are relatively ineffective in restoring transversion mutations at C:G pairs during hypermutation, or in restoring gene conversion in stably transfected DT40 cells. The results indicate that UNG does, indeed, act through uracil excision, but suggest that, in the presence of MSH2, efficient switch recombination requires base excision at only a small proportion of the AID-generated uracils in the S region. Interestingly, enforced expression of thymine-DNA glycosylase (which can excise U from U:G mispairs) does not (unlike enforced UNG or SMUG1 expression) potentiate efficient switching, which is consistent with a need either for specific recruitment of the uracil-excision enzyme or for it to be active on single-stranded DNA.

Abbreviations used: AID, activation-induced deaminase; dG, deoxyguanosine; dU, deoxyuridine; SMUG, single-stranded monofunctional uracil DNA glycosylase; TDG, thymine-DNA glycosylase; UNG, uracil-N-glycolase.

J.M. Di Noia's present address is Institut de Recherches Cliniques de Montréal, H2W 1R7 Montreal, QC, Canada.


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