Requirement of Non-canonical Activity of Uracil DNA Glycosylase for Class Switch Recombination

Abstract

Activation-induced cytidine deaminase (AID) and uracil DNA glycosylase (UNG) are required for class switch recombination (CSR). AID is involved in the DNA cleavage step of CSR, but the precise role of UNG is not yet understood. Mutations and deletions are footprints of abortive DNA cleavage in the immunoglobulin switch region in splenic B cells stimulated to undergo CSR. However, a UNG deficiency did not reduce the number of such footprints, indicating UNG is dispensable for the DNA cleavage step. Mutagenesis experiments revealed that the role of UNG in CSR depends on its WXXF motif. This motif is also essential for the interaction of UNG with the HIV viral peptide Vpr, which recruits UNG to the HIV particle. Furthermore, exogenous Vpr had a dominant-negative effect on CSR. These results suggest that UNG is recruited to the CSR machinery through its WXXF motif by a Vpr-like host factor and plays a novel non-canonical role in a CSR step that follows DNA cleavage. Activation-induced cytidine deaminase (AID)3 is necessary and sufficient for two major immunoglobulin (Ig) gene alterations, somatic hypermutation (SHM), and class switch recombination (CSR), which take place in the germinal center by the antigen stimulation of B cells (1-3). SHM introduces point mutations in the variable (V) region of Ig genes, resulting in an increase of the antibody affinity to antigens when coupled with selection of B cells by a limited amount of antigen. In the SHM reaction, DNA is first cleaved by single strand nicks, and mutations are subsequently introduced by erroneous DNA repair (4).CSR replaces the expressed heavy-chain constant (C H ) region exons from C with other downstream C H exons to generate antibodies with unaltered antigen specificity but with altered effector functions. In CSR, single strand nicks are introduced into both strands of the switch (S) regions to generate staggered cleavage ends with 3Ј or 5Ј overhangs, which are repaired to form blunt ends by gap-filling DNA synthesis and/or exonucleolytic digestion (4, 5). Finally, the blunt ends of the S and other S regions are joined by the non-homologous end joining (NHEJ) repair machinery (6 -9), giving rise to a large deletion in the chromosome and a looped-out circle of deleted DNA (10 -12). During this repair process, mutations and deletions are often introduced into the rearranged S regions. Some cleavages in the S regions are abortive and repaired without CSR, but mutations and deletions nonetheless arise during their repair (13-15).A deficiency of uracil DNA glycosylase (UNG), which removes U from DNA and triggers the base excision repair pathway, causes a severe reduction of CSR activity, to 10% of wild type (16). Interestingly, UNG deficiency does not significantly affect the SHM frequency, unlike deficiencies of the mismatch repair enzymes, which reduce the SHM frequency to 20 -50% (17-19). However, UNG deficiency does bias the target base specificity of SHM toward GC, as does a deficiency of MSH2 (16,20). The phenotypes of the UNG deficiency in CSR and SHM...

Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

“…The N terminus of UNG is unique in that it possesses proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) interacting sites (see Fig. S3), but the the region is not necessary for CSR (26,28). However, Guenzel et al (22) found that UNG-RPA interaction is necessary to suppress the mutation frequency in HIV-1, which is in contrast to what we observed in the case of AID-induced s-SHM.…”

“…Curiously, this recruitment is independent of its catalytic activity but dependent on the WxxF motif of UNG, which mediates interaction with the HIV-1 accessory protein Vpr. Interestingly, the WxxF motif was also found to be a critical site for CSR (26). Requirement of a noncanonical activity of UNG was proposed in AID-induced CSR (27,28), especially because no correlation could be found between CSR efficiency and catalytic activity of UNG.…”

“…1073/pnas.1402391111/-/DCSupplemental. drastically reduces CSR efficiency (26,28,30,32). If UNG is involved in AID-dependent DNA cleavage, UNG deficiency is supposed to reduce not only CSR but also SHM because SHM is not restricted C/G and depends on error-prone repair of DNA breakage (12).…”

Regulation of Abiotic Stress Response Through NtROS2a-mediated Demethylation in Tobacco

“…DNA glycosylases are subdivided into four groups according to their substrate specificity: excision of uracil, uracil-containing mismatches, alkylated bases and oxidized bases (Begum et al 2007). NtROS proteins are structurally related to Arabidopsis ROS1 and DME, which belong to a large family of uracil-containing mismatch DNA glycosylases.…”

“…Their induction profile upon abiotic stresses is indicative, as DNA glycosylases are considered to deal with oxidative damages, which are commonly caused by reactive oxygen species (Choi and Sano 2007;Begum et al 2007). The NtROS2a gene was induced expression after ABA, cold, salt and H 2 O 2 treatments.…”