Dicentric chromosome bridges exhibit impaired localization of Top2α and Rad17 to the bridge DNA next to the midbody. (A) Experimental procedure to study replication stress-induced DNA bridges (by HU), or dicentric chromosome bridges (induced by TC) in RPE-1 cells. (B) Schematic of a potential mechanism to generate dicentric chromosome bridges by sister chromatid fusion in RPE-1 cells after induction with TC. Deprotected tel sequences are shown in green. (C) FISH analysis of a chromatin bridge in TC-induced RPE-1 cells using a tel probe. A tel-positive signal on the DNA bridge is indicated by an arrow and shown at higher magnification. (D) Frequency of TC-induced chromatin bridges that are positive for cen or tel probes by FISH. Mean ± SD from three independent experiments (n > 60). (E and F) Chromatin bridges and frequency of chromatin bridges exhibiting DNA knots next to the midbody in HU-, TC-, or TC+HU-induced RPE-1 cells. Mean ± SD from three independent experiments (n > 100). (G–M and O–Q) Localization and mean intensity of GapR:GFP, Top2α, or Rad17 at the bridge DNA next to the midbody in HU-, TC-, or TC+HU-induced cells. Mean ± SD from n cells. Values in HU-induced cells were set to 1. Broken chromatin bridges are indicated by dotted arrows. Arrowheads indicate DNA knots. Midbodies (boxed areas) were reexported and are shown at higher magnification. (N) Percentage of broken DNA bridges HU-, TC-, or TC+HU-induced cells in the absence or presence of 300 nM VX-680 for 2 h. Mean ± SD from three independent experiments (n > 120). ***, P < 0.001 (ANOVA and Student’s t test). Numbers below/next to each bar indicate n. Scale bars, 5 μm.