6580 Nucleic Acids Research, 2011, Vol. 39, No. 15 Figure 6. Rad22-YFP foci are induced by the lacO-LacI RFB. (A) Rad22-YFP foci in strains with and without the lacO array with and without LacI (++) induction for 24 h. The data are mean values from three independent experiments with 100 cells assessed for foci in each experiment. Error bars represent standard deviations. (B) Example of Rad22-YFP focus co-localizing with lacO-LacI (+). (C) Co-localization of Rad22-YFP foci with the lacO array in the presence of repressed and induced levels of LacI (+). A total of between 200 and 300 Rad22-YFP foci-positive cells were assessed for co-localization in three independent experiments. The error bars are the standard deviations about the mean. (D) Images from time-lapse microscopy of Rad22-YFP foci (green) in a lacO array-containing cell undergoing anaphase after 24 h of LacI-eCFP (++) (blue) induction. (E) A comparison of the EBNA1 binds to a series of 20 repeated DNA elements known as the FRs within the viral origin of replication, and is required for the correct partitioning of the viral episomes in proliferating cells (43). An 800 bp DNA fragment containing 10 FR elements was inserted in place of the lacO array between the direct repeat of ade6 genes. Upon expression of the EBNA1 DNA binding domain from the nmt promoter in pREP41 a >700-fold increase in the frequency of Ade+ recombinants was observed (Figure 7D and Supplementary Table S3). Importantly >98% of these recombinants were deletion-types, which suggests that they stem from a similar set of problems as caused by the lacO-LacI repeat arrays. DISCUSSION It is now well established that the pausing of replication forks at RFBs can induce recombination, which can result in genome rearrangement. Recombination can be induced either through the remodelling of the stalled fork or by its cleavage to generate a DSB (11,14,44). In both cases the initiation of recombination is within replicated DNA (i.e. behind the replication fork), which makes accurate sister chromatid recombination possible and with promoting factors such as cohesin this becomes the predominant mechanism. It is thought that genome rearrangement only occurs when HR acts inadvertently between ectopic sites or low ﬁdelity repair mechanisms such as NHEJ or SSA are used in its stead (13). However, as discussed below, our study shows how a RFB can give rise to a DSB that is predisposed to cause genome rearrangement most probably by being formed in unreplicated DNA and therefore only repairable by low ﬁdelity mechanisms. DSB formation at a non-programmed protein–DNA RFB To date studies that have addressed the effect of protein– DNA RFBs on recombination in eukaryotes have focused on the programmed barriers RTS1 and the Fob1- dependent RFB from the rDNA array of S. cerevisiae (45). These have shown that such barriers can act as re- combination hotspots, and in some cases this may be due to DNA breakage, although recent data shows that most recombination at the RTS1 RFB stems from template ex- change events during replication restart rather than from DSB repair (5,6,14,46,47). Where DNA breakage has been observed it has generally been attributed to the action of speciﬁc nucleases, such as Slx1–Slx4 or Mus81–Eme1, cleaving the stalled fork. For example, replication fork breakage and Rad52/Rad22-dependent recombination in Figure 6. Continued percentage of cells with and without lacO/LacI (++) that undergo mitosis with one or two Rad22-YFP foci present throughout. The data are means from three independent experiments with 50–100 time-lapse movies of cells undergoing mitosis assessed in each experi- ment. Error bars represent standard deviations. (F) A comparison of the percentage of non-septated binucleate cells with and without a lacO array and with and without a UFB (each n = 50) that contain at least one Rad22-YFP focus after 24 h of LacI (++) induction. P-values were calculated by Fisher’s exact test (two-tailed).