Schematic and model for defining the outcome of sister chromatid alignment versus separation. (A) Use of halogenated thymidine analogs to distinguish between sister chromatids during S phase. RPE-1 and U2OS cells are incubated with 1 µM CldU for 24 h (first round of replication), chased with fresh media for 1 h, and incubated with 200 µM IdU for 30 min (second round of replication). (B) Predicted outcomes for sister chromatid alignment (left) versus separation (right). If sister chromatids remain aligned/adjacent to each other (left), they will result in DNA fibers that are red–green–red if there is no breakage, breakage at a, or breakage at b, and in fibers that are red–green upon breakage at both at a and b. In contrast, if the sister chromatids become separated (right), they will result in DNA fibers that are red–green–red if there is no breakage, red–green upon breakage at a, red–green–red and green only upon breakage at b, and red–green and green only upon breakage at both a and b.