Inhibition of UNG2 outside S phase blocks Ig class switching. (A) Flow cytometric analysis of cell cycle in transduced LPS-activated B cell blasts. Contour plots show BrdU incorporation as a function of DNA content in gated GFP⁺ cells that were fixed, permeabilized, and stained after a 1-h BrdU pulse (this experiment was performed twice). The fraction of total GFP⁺ cells encompassed by each gate is indicated. Bottom left gate, G1 phase cells; top gate, S phase cells; bottom right gate, G2/M phase cells. The histogram (bottom right) shows distribution of DNA content in GFP⁺ cells expressing ugi-GFP (solid gray), ugi-GFP-rag (blue), or ugi-GFP-cyc (red). The vertical scales are arbitrarily adjusted to mimic Fig. 1 C. (B) The distribution of DNA content in GFP⁺ LPS-activated B cells transduced to express GFP (solid gray), GFP-rag (blue), GFP-cyc (red; top), or ugi-GFP-cyc (bottom) or ugi-GFP-cyc^Dmut (solid pink) fusion proteins. The vertical scales are arbitrarily adjusted to mimic Fig. 1 C. (C) Switching from surface IgM to IgG1 expression in transduced B cell blasts. C57BL/6 splenocytes were cultured overnight with bacterial LPS, transduced to express the fusion proteins indicated, and then placed back into culture with LPS+IL-4. 3 d later, cells were surface labeled with APC-conjugated anti-IgG1 antibody. The frequencies of IgG1⁺ cells in the GFP⁺ populations, as determined by flow cytometry, are indicated for four independent experiments (some viruses were tested <4 times). (D) U excision activity in GFP⁺ transduced B cell blasts. 2 d after transduction, whole-cell extracts were prepared from sorted GFP⁺ B cells activated with LPS. (top) Cleavage of a fluorescent U-bearing oligonucleotide (heteroduplexed to create a U:G base pair, see Table S1) by extracts equivalent to 2 × 10⁴ sorted cells (supplemented with 5 units of recombinant APE1 [New England Biolabs] and with anti-SMUG1 antibody PSM1) in a 4-h incubation at 37°C. (bottom) Western blot for tubulin in the same cell extracts. A repeat experiment produced the same results (not depicted). (E) Switching to IgG₁ as a function of cell division number in one experiment. Splenocytes were labeled with CellTrace Violet before culture and transduction. Inset shows the distribution of CellTrace Violet fluorescence for all cells (gray), or GFP^+ve cells (thick line) expressing ugi-GFP-rag. The number of divisions each cohort has undergone is indicated by the digits above each cohort peak. Plotted is the percentage of surface IgG₁⁺ cells detected in each division cohort of GFP⁺ cells transduced to express fusion proteins. Symbols as in Fig. 2 C. A repeat experiment produced the same results (not depicted). This experiment is one of the replicates shown in Fig. 2 C.