B cells from hyper-IgM patients carrying UNG mutations lack ability to remove uracil from ssDNA and have elevated genomic uracil

The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to...

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Bibliographic Details
Published in:The Journal of experimental medicine 2005-06, Vol.201 (12), p.2011-2021
Main Authors: Kavli, Bodil, Andersen, Sonja, Otterlei, Marit, Liabakk, Nina B, Imai, Kohsuke, Fischer, Alain, Durandy, Anne, Krokan, Hans E, Slupphaug, Geir
Format: Article
Language:English
Subjects:
B-Lymphocytes
Blotting, Western
Cell Line
Comet Assay
Cytidine Deaminase - immunology
DNA Glycosylases - genetics
DNA Glycosylases - physiology
DNA, Single-Stranded - metabolism
Escherichia coli
Humans
Hypergammaglobulinemia - genetics
Hypergammaglobulinemia - immunology
Immunoglobulin Class Switching - immunology
Immunoglobulin M - immunology
Immunoprecipitation
Microscopy, Confocal
Models, Immunological
Mutation - genetics
Protein Transport - physiology
Somatic Hypermutation, Immunoglobulin - genetics
Somatic Hypermutation, Immunoglobulin - immunology
Uracil - metabolism
Uracil-DNA Glycosidase
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Summary:The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to function as an mRNA editing deaminase or as a single-strand DNA deaminase. In the latter model, SHM may result from replicative incorporation of dAMP opposite U or from error-prone repair of U, whereas CSR may be triggered by strand breaks at abasic sites. Here, we demonstrate that extracts of UNG-proficient human B cell lines efficiently remove U from single-stranded DNA. In B cell lines from hyper-IgM patients carrying UNG mutations, the single-strand-specific uracil-DNA glycosylase, SMUG1, cannot complement this function. Moreover, the UNG mutations lead to increased accumulation of genomic uracil. One mutation results in an F251S substitution in the UNG catalytic domain. Although this UNG form was fully active and stable when expressed in Escherichia coli, it was mistargeted to mitochondria and degraded in mammalian cells. Our results may explain why SMUG1 cannot compensate the UNG2 deficiency in human B cells, and are fully consistent with the DNA deamination model that requires active nuclear UNG2. Based on our findings and recent information in the literature, we present an integrated model for the initiating steps in CSR.
ISSN:0022-1007
1540-9538
1892-1007
DOI:10.1084/jem.20050042