formation compared to the equivalent wild-type strain (Figure 5B). This is consistent with the idea that Pfh1 is needed at other genomic sites to aid their replication and thereby avoid bridge formation. It is thought that unresolved UFBs in human cells may account for at least some of the DNA breakage that occurs at common fragile sites (19). If DNA breakage in the lacO array is similarly associated with a failure to properly resolve an anaphase bridge at this site then mutants such as pfh1-mt*, which manifest a greater fre- quency of bridges, should display more DSBs and conse- quently more recombination. With LacI (++) there is no difference in the frequency of lacO-LacI-induced Ade+ re- combinant formation between wild-type and pfh1-mt* strains (data not shown). However, this is expected because as mentioned earlier recombinant formation is already at or near saturating levels in the wild-type. With LacI (+) a 3-fold increase in the frequency of lacO- LacI-induced Ade+ recombinants is seen in the pfh1-mt* mutant, which is consistent with Pfh1 being required to overcome the RFB and thereby avert anaphase bridge formation and associated DNA breakage (Figure 5F and Supplementary Table S1). Evidence that the cell mounts a response to RFBs that continues through mitosis The discovery of UFBs in human cells has revealed that replication intermediates frequently persist into mitosis, and cause the deployment of repair proteins, which act even as late as anaphase to resolve them (18,19). To see if the lacO-LacI RFB provokes a similar response we monitored sub-nuclear foci of Rad22 [fused to yellow ﬂuorescent protein (YFP)]—a protein that plays a key role in promoting replication restart at the RTS1 RFB (5). In asynchronously growing populations of cells the presence of the lacO array together with LacI (++) results in a 3-fold increase in the number of cells with one or more foci (P < 0.01) (Figure 6A). A signiﬁcant increase in the percentage of cells with foci was not seen with LacI (+) (data not shown), however increased co-localization of Rad22-YFP foci with the lacO array was (Figure 6B and C). Together these data show that the lacO-LacI RFB provokes a response that involves Rad22. At least some of the Rad22 foci caused by the lacO-LacI RFB are likely to be responding to the DNA DSB that is formed in the lacO array. As discussed above these DSBs should be repaired prior to mitosis. If this assumption is correct then Rad22 foci that persist into mitosis are likely to indicate the presence of unresolved replication inter- mediates at the lacO-LacI RFB. To investigate this we monitored foci in live cells by time-lapse microscopy (e.g. Figure 6D). Rad22-YFP foci are observed in only 2% of mitoses in the absence of LacI and 5% when LacI (++) is present in strains without the lacO array (Figure 6E). However, in strains with the lacO array LacI (++) expression results in an almost 3-fold increase in the number of mitoses with Rad22-YFP foci (P < 0.005) (Figure 6E). Typically a single Rad22-YFP focus is observed, which segregates with one DNA mass during Nucleic Acids Research, 2011, Vol. 39, No. 15 6579 anaphase (e.g. Figure 6D). In 60% of cases a second Rad22-YFP focus then appears, which is associated with the other DNA mass, and the two foci sometimes adopt a conﬁguration where they reside at either end of an anaphase bridge (Figure 6D), which is reminiscent of the pattern of FANCD2 immunostaining observed at UFBs in human cells (19). Further analysis of ﬁxed cells conﬁrms that Rad22-YFP foci are more prevalent in non-septated binucleate cells containing a lacO-LacI-induced UFB than in the equivalent cells without a UFB (Figure 6F). Altogether these data suggest that Rad22 is recruited to replication forks that are blocked at the lacO-LacI array to help promote their restart, and that this response con- tinues even during anaphase. Shorter arrays of lacO induce UFBs and recombination The 115 repeat lacO array was chosen because we wanted to impose a very potent barrier to replication. However, natural protein–DNA barriers are unlikely to be composed of so many adjacent protein–DNA ‘units’, and therefore to investigate whether shorter lacO arrays could generate similar problems we made a series of strains containing between 2 and 21 repeats in place of the 115 repeat array. With LacI (+) expression, no signiﬁcant increase in direct repeat recombination was seen in the strain with two lacO repeats (Figure 7A and Supplementary Table S2). However, with 12, 15 and 21 lacO repeats a 5- to 8-fold increase in direct repeat recombinantion was observed with 90% or more of the recombinants being deletion- types (Figure 7A and Supplementary Table S2). With LacI (++) expression, two lacO repeats are sufﬁcient to cause a 10-fold increase in recombinants compared to a strain with no repeats, and 12–21 repeats induce near sat- urating levels of recombinants similar to the 115 repeat array (Figure 7B and Supplementary Table S2). Again the majority of the induced recombinants are deletion- types, from 80% with two repeats to >95% with 12–115 repeats (Figure 7B and Supplementary Table S2). Similar to the 115 repeat array, the high frequency of re- combinants seen with the shorter repeat arrays correlates with increased levels of UFB formation (Figure 7C and data not shown). Altogether these data indicate that the pathologies associated with problems in replicating the 115 repeat lacO-LacI array are also manifested by shorter arrays, albeit with reduced frequency especially when the array consists of only two repeats or LacI is expressed at the lower level. EBNA1 binding to the FRs induces recombination like lacO-LacI The interaction between LacI and lacO is similar to that of many transcription factors with their cognate DNA binding sites. However, to conﬁrm that there was nothing unusual about the interaction between lacO and LacI that made it an especially potent RFB, we tested whether DNA binding by an unrelated protein would cause the same telltale increase in direct repeat recombination. The protein that we chose was EBNA1, which is one of only a few proteins that are expressed by the Epstein-Barr virus (EBV) during its latent infection of B lymphocytes (42).