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 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.
Kavli
Bodil
B
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
Andersen
Sonja
S
Otterlei
Marit
M
Liabakk
Nina B
NB
Imai
Kohsuke
K
Fischer
Alain
A
Durandy
Anne
A
Krokan
Hans E
HE
Slupphaug
Geir
G
D001402:B-Lymphocytes; D015153:Blotting, Western; D002460:Cell Line; D020552:Comet Assay; D003564:Cytidine Deaminase / Q000276:immunology; D045647:DNA Glycosylases / Q000235:genetics* / Q000502:physiology; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006801:Humans; D006942:Hypergammaglobulinemia / Q000235:genetics / Q000276:immunology*; D017578:Immunoglobulin Class Switching / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology*; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D018448:Models, Immunological; D009154:Mutation / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics / Q000276:immunology; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
D004277:DNA, Single-Stranded; D007075:Immunoglobulin M; D014498:Uracil; C492570:CCNO protein, human; D045647:DNA Glycosylases; C498095:SMUG1 protein, human; D051981:Uracil-DNA Glycosidase; D003564:Cytidine Deaminase