Uracil-qPCR detects uracil accumulation at the antibody 5′-Sμ region. (A) Diagram of how Pfu polymerase and its mutant Pfu-Cx act on DNA. Uracil-free DNA can be amplified by both Pfu and Pfu-Cx polymerases (left). The lack of uracil recognition by Pfu-Cx polymerase allows it to replicate uracil-containing DNA, whereas Pfu polymerase stalls (right). This differential ability to amplify uracil-containing DNA is harnessed to measure the relative level of uracil (see Materials and methods; Horváth and Vértessy, 2010). (B) Experimental layout. In vitro–activated B lymphocytes were arrested with nocodazole 42 h after stimulation. 6 h later, nocodazole was removed and B cells were collected for analysis at different time points. (C) DAPI staining for DNA content at the indicated time points upon nocodazole release. Red, the sorted populations for experiments in Fig. 2 D and Fig. S2 (E and F). Upon release, B cells were still mainly in M phase at 0.5 h, whereas the released G1 population increased to ∼40% at 1.5 h (early G1) and 60% at 4 h (middle G1). At 6 h, some cells entered S phase, and 30% were still in G1 (late G1). (D) Relative uracil levels at the antibody 5′-Sμ region, as determined by Uracil-qPCR (see Materials and methods). AID-proficient and -deficient cells are compared. Error bars, SEM; *, P < 0.01, two-tailed Student t test. Three independent experiments are shown. (E) Diagram of the sorting strategy for the analysis of G1, S, G2, and M cells. In vitro–activated B cells at different cell cycle stages were sorted according to phospho-Histone H3 (Ser10) and DNA content (DAPI). (F) The relative uracil levels at 5′-Sμ at different cell cycle stages were determined by Uracil-qPCR in WT and UNG-deficient B cells. One experiment is shown.