this strain (Figure 4E). This indicates that the majority of the DSBs formed in an rqh1D exo1D double mutant are repaired successfully, albeit their higher accumulation suggests that the kinetics of repair may be slower than in wild-type. Furthermore, the low level of Ade+ recom- binants suggests that SSA between the ade6 repeats is not the main repair mechanism (Figure 4F). Instead we suspect that repair occurs mostly by annealing between the lacO repeats. The larger reduction in viability ( 40%) observed in rad22D and rad22D swi10D mutants compared to the rqh1D exo1D double mutant suggests that this alternative means of repair is at least partly dependent on Rad22 (Figure 4E). Altogether these data show that genes required for SSA are also needed for the efﬁcient repair of DSBs induced in the lacO array following LacI (++) induction. lacO-LacI induces the formation of structures analogous to UFBs Whilst monitoring the expression of LacI-eCFP by epi- ﬂuorescence microscopy we observed that 15% of cells undergoing mitosis display a bridge of LacI-eCFP ﬂuor- escence spanning the interval between the segregating DNA masses (Figure 5A and B). Unlike classical anaphase bridges, the bridges highlighted by LacI-eCFP ﬂuores- cence are generally undetectable by DNA speciﬁc dyes such as SYBR Green and DAPI (Figure 5A and data not shown). Like other fungi, S. pombe undergoes a closed mitosis where the nuclear membrane does not break down, and therefore the bridge of ﬂuorescence could simply represent a tendril of nucleoplasm devoid of DNA. However, Histone H3.2 fused to GFP localizes to the bridges (Figure 5C) suggesting that they contain chromatin, and more importantly they are only prevalent in wild-type cells that contain the lacO array (Figure 5B), which indicates that the interaction between LacI and lacO is necessary for their formation. Furthermore, we observe lacO-LacI-dependent bridges that persist until the formation of the cell division septum suggesting that unresolved anaphase bridges could be broken during cyto- kinesis thereby providing a mechanism for the formation of DSBs in the lacO array (Figure 5D). In humans there is a class of anaphase bridges that are detectable by immunostaining for DNA repair proteins such as BLM and FANCD2 but undetectable by DNA stains (18,19). Intriguingly these so-called UFBs are fre- quently associated with common fragile site loci including FRA3B, FRA16D and FRA7H, especially following in- hibition of replication by aphidicolin, and are thought to be caused by incomplete replication within these regions (19). We propose that the lacO-LacI-dependent bridges are analogous to UFBs and therefore will refer to them by this name henceforth. lacO-LacI UFBs are not caused by unrepaired DNA DSBs In budding yeast, a single DSB induces a checkpoint- mediated arrest of the cell cycle. However, after 6–8 h cells adapt to the checkpoint and undergo mitosis even if the DSB is unrepaired, which in turn can impede Nucleic Acids Research, 2011, Vol. 39, No. 15 6577 proper chromosome segregation (38). In our case DSBs at the lacO array should be readily repaired by SSA during a checkpoint-mediated cell cycle arrest because the ade6 repeats are close enough to the array for sufﬁcient DNA resection to occur prior to adaptation (38). Nevertheless, it was possible that unrepaired DSBs at the lacO array were responsible for the bridges that we had detected. If true then they should increase in frequency if the checkpoint response to the DSB is prevented as this would result in more unrepaired DSBs persisting into mitosis. Consistent with the DSB inducing a checkpoint response 33% of cells containing the lacO array in an asynchronously growing population are longer than 16 mm after a 24 h induction of LacI (++) compared to 8% without the lacO array (Figure 5E). This subpopulation of long cells is absent in the equivalent chk1D mutant strain indicating that lacO-LacI induces a Chk1-dependent cell cycle arrest (Figure 5E). Importantly, deletion of chk1 does not result in a greater frequency of lacO-LacI (++)-dependent bridges indicating that unrepaired DSBs are unlikely to be their cause (Figure 5B). lacO-LacI UFBs are not caused by unresolved recombination intermediates The UFBs detected by immunostaining for FANCD2 in human cells increase in frequency when Rad51 is depleted indicating that bridges are unlikely to be due to unresolved recombination intermediates between sister chromatids (19). Similarly lacO-LacI-dependent bridges increase in fre- quency in a rad22D rad51D double mutant (Figure 5B). Interestingly the frequency of bridges also increases in rad22D rad51D cells expressing LacI (++) without the lacO array compared to the equivalent wild-type strain (Figure 5B). This suggests that HR may play a role at other genomic loci to prevent the formation of UFBs caused by LacI binding to DNA non-speciﬁcally or other replication fork impediments. In the latter case, LacI-eCFP would simply highlight the presence of the UFB by occupying the nucleoplasm in the bridge. Pfh1 prevents lacO-LacI-dependent UFB formation and hyper-recombination In budding yeast non-nucleosomal protein–DNA complexes that could impede replication fork progression are removed by the putative ‘sweepase’ activity of the Rrm3 DNA helicase (39,40). If lacO-LacI-dependent UFBs are caused by replication fork stalling at protein– DNA barriers then they should increase in frequency in cells lacking the S. pombe orthologue of Rrm3, Pfh1 (41). Pfh1 has an essential role in the mitochondria of S. pombe and therefore we made use of a mutant pfh1 gene (pfh1-mt*) that expresses protein that localizes to the mitochondria but not the nucleus (41). A pfh1-mt* strain containing both lacO and LacI (++) shows a 2-fold increase in UFBs compared to wild-type (Figure 5B), which is consistent with the idea that bridge formation stems from an inability of replication forks to overcome the lacO-LacI barrier. Like in the rad22D rad51D double mutant LacI (++) expression in the pfh1-mt* mutant without a lacO array also results in an increase in bridge