Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2005 Jun 20;201(12):2011-21.
doi: 10.1084/jem.20050042.

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

Bodil Kavli  1 , Sonja Andersen, Marit Otterlei, Nina B Liabakk, Kohsuke Imai, Alain Fischer, Anne Durandy, Hans E Krokan, Geir Slupphaug
Affiliations
Comparative Study

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

Bodil Kavli et al. J Exp Med. .

Abstract

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
UNG2 is the major uracil-DNA glycosylase in B cell nuclei. (A) Immunoprecipitation and Western blot analysis of UNG and SMUG1 in total and nuclear extracts prepared from the UNG proficient control LCL. (B) Total specific UDG activity in UNG-proficient LCL nuclear extracts measured by high turnover activity assays using 36 pmol (1.8 μM) [3H]-dUMP labeled calf thymus DNA substrates (gray bars: double-stranded substrate; black bars: single-stranded substrate). Extracts (2 μg protein) were pretreated with 0.5 μg preimmune IgGs (0-Ab), neutralizing anti-hUNG (PU101), or neutralizing anti-hSMUG1 (PSM1) IgGs, or both neutralizing IgGs. (C) UDG activities in UNG-proficient LCL nuclear extract measured by low turnover activity assays using 0.2 pmol (0.02 μM) [33P]-labeled 19-mer deoxyoligonucleotide with uracil in U:A, U:G, or Uss contexts. The samples in lane 1–4 were pretreated as in (A). Lane 5: nuclear extract was preincubated with 10 ng Ugi. Lane 6: samples were treated as in lane 4. In addition, 200 ng purified UNG2 was included in the reaction. Lane 7: samples were treated as in lane 4. In addition, 200 ng purified SMUG1 was added to the reaction. Note that two product bands appear to be formed consistently. The lower of these bands represents the 9-bp product formed by cleavage of the AP site by hot piperidine. The upper band represents a 9-bp product in which the AP site has been cleaved by AP endonuclease (present in the extracts), before the addition of hot piperidine. Such cleavage results in loss of the 3′-phosphate and slower migration during PAGE. (D) UDG activity of purified, recombinant UNG2 and SMUG1 in the presence of 0.2 pmol purified, recombinant APE1 was measured using 0.2 pmol [33P]-labeled 19-mer deoxyoligonucleotide with uracil in U:A, U:G, or Uss as substrate. Lane 1: 0 pmol UNG2/SMUG1; lane 2: 0.2 fmol UNG2/SMUG1; lane 3: 2 fmol UNG2/SMUG1; lane 4: 20 fmol UNG2/SMUG1; lane 5: 0.2 pmol UNG2/SMUG1; and lane 6: 2.0 pmol UNG2/SMUG1.
Figure 2.
Figure 2.
UNG2-F251S is fully active and stable in vitro, but the mutation impairs expression of active protein in B cells. (A) Overall structure of the WT UNG complexed with a uracil-containing DNA duplex, with F251 highlighted. The close-ups show that F251 is surrounded by several hydrophobic side chains, and that the F251S substitution results in a “hole” in the central hydrophobic region of UNG. (B) P2 nuclear extract analyzed using 0.2 pmol (0.02 μM) [33P]- labeled 19-mer deoxyoligonucleotide with uracil in U:A, U:G, or Uss contexts. The samples were pretreated as in Fig 1 B. (C) Temperature optimum (left panel) and thermal stability (right panel) of purified, recombinant UNG2-WT and UNG2-F251S measured by high turnover activity assays using 36 pmol (1.8 μM) [3H]-dUMP labeled calf thymus DNA substrates. (D, left) Immunoprecipitation and Western blot analysis of purified, recombinant UNG2-WT and UNG2-F251S, demonstrating that the PU101 antibody precipitates the WT and mutant proteins with equal efficiency. (right) IP and Western blot analysis of UNG (using PU101) and SMUG1 (using PSM1) in whole cell extracts from P2 (UNG2-F251S) and C1 (control 1). (E) Trace levels of UNG activity in P2 nuclear (gray bars) and cytosolic + mitochondrial (black bars) fractions measured by high turnover activity assays using 36 pmol [3H]-dUMP [3H]-labeled heat-denatured calf thymus DNA substrate under high sensitivity assay conditions (4 μg protein extracts, 30 min incubation at 37°C). Note the higher resolution of the y-axis compared with Fig. 1 B.
Figure 3.
Figure 3.
UNG2-F251S is abnormally translocated to mitochondria. (A) WT UNG2-EYFP or UNG2-F251S-EYFP transfected in HeLa cells and followed in live cells for 2 d. (B) Control coexpression of WT UNG2 with different fluorescent tags (upper row). UNG2-F251S and WT UNG2 are localized different in human cells, demonstrated by coexpression of UNG2-F251S-EYFP and WT UNG2-ECFP in the same cell (lower row). (C) Coexpression of WT UNG1-ECFP and UNG2-EYFP demonstrates distinct sorting to mitochondria and nuclei, respectively (upper row), whereas coexpression of mutant UNG2-F251S-EYFP and UNG1-ECFP abolishes translocation of the mutant to nuclei, while increasing its accumulation at mitochondria. (D) UNG2-F251S-EYFP but not WT UNG2-EYFP colocalizes with mitochondria in fixed cells as demonstrated using an antibody against human mitochondria.
Figure 4.
Figure 4.
Steady-state genomic uracil levels are increased in LCLs from UNG mutant patients; thus, SMUG1 or other UDGs cannot compensate for UNG deficiency. (A) Single cell gel electrophoresis (comet assay) of alkali-treated cells. Each of the comets was assigned a score for the level of apparent DNA damage (given as arbitrary units, see Materials and methods). Cells were treated with recombinant UNGΔ84 before electrophoresis (black bars) or with buffer alone (open bars). Error bars represent the standard deviation calculated from four independent experiments (each in duplicate). Immunoprecipitation and Western blot analysis of UNG and SMUG1 from the whole cell LCL lysates are shown below. (B) Photomicrographs illustrating results from the comet assay. C1 and UNG mutant P3 cells are representative for all cell lines in the respective groups. (C) SMUG1 or other UDG activities are not up-regulated in UNG-deficient LCLs. Nuclear extracts (2 μg protein) from UNG-proficient (C1, C2) and UNG-deficient (P1, P2, P3) LCLs were preincubated with 10 ng Ugi and subjected to analysis using low turnover activity assays using 0.2 pmol (0.02 μM) [33P]-labeled19-mer deoxyoligonucleotide with uracil in U:A, U:G, or Uss contexts. In addition, either 0.5 μg preimmune IgGs (0-Ab) or neutralizing anti-hSMUG1 IgGs (SMUG1-Ab) were added to the extracts.
Figure 5.
Figure 5.
Integrated model for initiation of CSR. Deamination of deoxycytidine in ssDNA close to the transcription complex initiates MutSα-independent (left) and MutSα-dependent (middle and right) pathways. The MutSα-independent pathway requires UNG2 that creates a large number of AP sites in both strands. After reannealing, opposing or closely spaced AP sites directly generate DSBs by AP-endonuclease (APE1) cleavage. When the cellular level (or function) of UNG2 is partially impaired, fewer AP sites are generated, and may not sustain DSB formation by direct cleavage. However, even a small number of AP sites generated by UNG2 would be sufficient to create nicks recognized by DNA mismatch repair factors and to initiate 5′-3′ processing toward MutSα bound to unprocessed U:G mismatches. If UNG2 is impaired fully, the MutSα-dependent pathway still may operate—although with strongly reduced efficiency—by using alternative nicking factors, such as ERCC1/XPF or TDG/NEIL1/SMUG1, followed by APE1. In such a scenario, the different pathways are likely to function in parallel, and the speed at which individual pathways operate may depend on the availability of the various factors at any given time point.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Krokan, H.E., H. Nilsen, F. Skorpen, M. Otterlei, and G. Slupphaug. 2000. Base excision repair of DNA in mammalian cells. FEBS Lett. 476:73–77. - PubMed
    1. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 102:553–563. - PubMed
    1. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al. 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell. 102:565–575. - PubMed
    1. Honjo, T., M. Muramatsu, and S. Fagarasan. 2004. AID: how does it aid antibody diversity? Immunity. 20:659–668. - PubMed
    1. Muramatsu, M., V.S. Sankaranand, S. Anant, M. Sugai, K. Kinoshita, N.O. Davidson, and T. Honjo. 1999. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J. Biol. Chem. 274:18470–18476. - PubMed

Publication types

MeSH terms