(A) S. pombe strains harbouring the site-specific replication stall system RuraR. Replication fork stalling at RTS1 is induced in the absence of thiamine (on). Plates were incubated at 30°C for 3 days. Unlike the HR-defective rad51Δ strain, smc6 hypomorphs do not lose viability on induction of replication stalling at RTS1. (B) PCR-based assay for translocation between RTS1 at RuraR and the native RTS1 at the mating type locus in ura4− colonies generated in the ura4 loss of gene function assay (Lambert et al., 2005). Left: Schematic to show the three primer pairs used. One pair (red arrows) amplifies the junction resulting from ectopic recombination between chromosome II and III (TLII/III). The second pair (grey arrows) amplifies the ura4 locus to distinguish point mutations, truncations (internal deletions), and full-length deletions. rng3 (blue arrows), an essential gene located between RuraR and the telomere, is amplified as positive control. Right: Example of control PCRs (top) and PCRs of 5′-fluoroorotic acid (5-FOA) resistant/ura4− colonies (bottom). The rng3 product is amplified in all strains, but not in the negative control (‘-'). ura4 is amplified only in a RuraR strain, but not in wild type (wt) (harbours full deletion of ura4, ura4-D18), the translocation positive control (‘+', gift from S. Lambert [Lambert et al., 2005]) or the negative control. Translocation between chromosome II and III can only be detected in the positive control. (C) PCR assay results for ura- colonies of smc6+, smc6-74 and smc6-X derived from the RuraR ura4 loss assay carried out in the presence (RuraR arrest ‘Off’) or absence (RuraR arrest ‘On’) of thiamine.