Chromatin bridges but not UFBs are sensed by the DNA damage checkpoint. (A) Ddc2 binds to DNA in anaphase bridges. Cells (VS34-1A) coexpressing Ddc2-YFP and Dpb11-CFP were analyzed. (B) Sml1 levels decrease in response to Rad52-bound chromatin bridges. Cells (SMG260-7C) expressing YFP-Sml1, Dpb11-CFP, and Rad52-RFP were analyzed as in A. (C) Chromatin bridges but not UFBs activate the DNA damage response. Hoechst staining and YFP-Sml1 levels were quantified in cells from B with Rad52-positive and Rad52-negative Dpb11 bridges. (D) Dpb11 stabilizes long UFBs. Cells (ML734-9B) coexpressing RFP-NLS, Ina1-CFP, and Spc110-CFP were grown overnight with or without 20 µg/ml doxycycline to repress Dpb11-YFP expression from the Tet-Off (tetO₂) promoter and imaged after Hoechst staining. The length of UFBs (Hoechst-negative RFP bridges; distance between SPBs) was measured in 20 cells for each condition. Error bars represent 95% confidence intervals. (E) The NoCut checkpoint senses Dpb11 bridges. Wild-type (ML734-9B), ahc1Δ (ML737-3C), top2-1 (ML734-11D), ahc1Δ top2-1 (ML737-11A), slk19Δ (ML735-1C), and slk19Δ top2-1 (ML735-13A) cells expressing Dpb11-YFP, NLS-RFP, Spc110-CFP, and the plasma membrane marked by Ina1-CFP were grown overnight with or without 20 µg/ml doxycycline (Dox) to repress Dpb11 expression and imaged after Hoechst staining. The abscission index was calculated as the ratio of cells with contracted versus resolved plasma membrane as described previously (Mendoza et al., 2009). The NLS-RFP marker was used to estimate the frequency of UFBs in the Dpb11-depleted cells. For each condition, two independent experiments were performed to examine abscission in 30–78 anaphase cells. *, †, and ‡ indicate significant difference from wild-type, untreated, and top2-1, respectively (P < 0.05, χ² test).