Uracil-DNA glycosylase is not implicated in the choice of the DNA repair pathway during B-cell class switch recombination

Abstract

y Mature B-cells express membrane IgM and IgD (of same specificity) through alternative splicing of a pre-mRNA encompassing constant (C) μ and C δ genes. After encountering antigen, Bcells undergo class switch recombination (CSR) that substitutes the C μ gene with C γ , C ε , or C α , thereby generating IgG, IgE, and IgA antibodies with the same antigenic specificity but new effector functions. DNA-editing enzyme activation-induced deaminase (AID) is essential for CSR by targeting switch (S) regions preceding C μ (namely, the S μ donor region) and the C γ , C ε , and C α genes (namely, the S γ,ε,α acceptor regions). 1, 2 CSR is controlled in cis by IgH locus super-enhancers 3 and in trans by a wide spectrum of enzymes and proteins. 1, 2 Among them, the role of the uracil DNA glycosylase (UNG) remains controversial. UNG is a key enzyme of base excision repair, which carries out faithful repair. Some authors estimate that during CSR, the UNG enzymatic activity removes the AID-induced dC to dU converted base of singlestrand DNA, generating abasic sites and leading to DNA strand breaks. 1 For other authors, the role of UNG is to stabilize the S-S synapse and to recruit DNA repair factors that facilitate the endjoining process. 4,5 Thus, the classical non-homogenous end joining pathway would be increased over the alternative end joining (A-EJ) pathway in UNG-deficient mice, 4 suggesting an intriguing role of UNG in promoting the A-EJ pathway. These results were based on the analysis of several S μ -S γ1 sequences obtained in UNG-deficient conditions due to technical limitations (conventional Sanger sequencing) to investigate S-S junction molecular signatures. We recently reported a new computational tool (CSReport) for automatic analysis of CSR junctions sequenced by high-throughput sequencing. 6 We used this tool to analyze the rare S μ -σ δ junctions formed during IgD CSR 7, 8 and S μ -S γ3 , S μ -S γ1 , and S μ -S α junctions in wild-type (wt) mice. 9 We thus used CSReport and high-throughput sequencing to analyze the molecular signature of S μ -S γ3 , S μ -S γ1 , and S μ -S α junctions in UNG-deficient mice in detail.Our research has been approved by our local ethics committee review board (Comité Régional d'Ethique sur l'Expérimentation Animale du Limousin, Limoges, France) and carried out according to the European guidelines for animal experimentation. Wt mice and UNG-deficient mice (a gift from Dr. Tomas Lindahl, UK) were used. Single-cell suspensions of spleen cells were cultured for 4 days at 1 × 10 6 cells/ml in RPMI 1640 with 10% fetal calf serum and 5 μg/ml LPS, with (CSR toward IgG 1 ) or without (CSR toward IgG 3 ) addition of 20 ng/ml IL-4 or 2 ng/ml TGFβ (PeproTech, Rocky Hill, NJ) (CSR toward IgA). 3,9,10 Splenocytes were washed in PBS and stained with various antibodies: anti-B220-PC5, anti-CD138-