(A) Schematic showing the position of the Ter2/3 replication fork barrier downstream of RTS1-AO on chromosome 3. For the live cell imaging experiments, in B) and C), a lacO array is also inserted downstream of RTS1-AO to enable tracking of the locus, in cells expressing LacI-tdKatushka2, by time-lapse fluorescent microscopy. Rad52 is tagged with yellow fluorescent protein (YFP) and forms nuclear foci that mark sites of recombination activity. Co-localization of a Rad52-YFP focus with a lacO-LacI-tdKatushka2 focus indicates recombination activity at RTS1 (Nguyen et al., 2015). (B) Representative stills taken from a time-lapse movie of a wild-type cell showing a pair of daughter cells (outlined by white dashed lines) each with a Rad52-YFP focus. In the right-hand cell the Rad52-YFP focus co-localizes with a lacO-LacI-tdKatushka2 focus that marks the position of RTS1-AO in the nucleus. The scale bar represents 1 µm. (C) The effect of Ter2/3 on the percentage of cells with a Rad52-YFP focus co-localizing with a lacO-LacI-tdKatushka2 focus in the first 90 min period post-anaphase. S-phase starts 10–15 min after anaphase (Nguyen et al., 2015). Delaying the incoming fork, by inserting a Ter2/3 barrier downstream of RTS1-AO, provides more time for Rad52 recruitment to the RF blocked at RTS1, as evidenced by the greater percentage of cells with a Rad52-YFP focus co-localizing with a lacO-LacI-tdKatushka2 focus. This result indicates that RF convergence and termination at RTS1-AO prevents Rad52 recruitment, and agrees with the finding of a previous experiment where incoming replication was delayed by deleting ori1253 (Nguyen et al., 2015). The strain with no Ter2/3 is MCW6556 (n = 123) and the strain with Ter2/3 is MCW7304 (n = 44).