ssDNA tail by an exonuclease (10). A duplex DNA end can also be generated by structure-speciﬁc nucleases such as Mus81-Eme1 cleaving the stalled fork (11). In all of the cases above one sister chromatid is essentially left intact and therefore can act as the donor DNA mol- ecule during HR. Indeed it has been shown that accurate sister chromatid recombination, which is catalysed by Rad51, is the predominant pathway for repairing broken replication forks (12). In the absence of Rad51 repair proceeds by alternative low-ﬁdelity mechanisms such as single-strand annealing (SSA) or non-homologous end joining (NHEJ) (13). Much of our current understanding of how protein– DNA RFBs can induce HR in eukaryotes has come from recent studies of a site-speciﬁc RFB in the ﬁssion yeast Schizosaccharomyces pombe called RTS1 (5,6,14,15). This programmed RFB has evolved to work closely with the replisome and components of the so-called fork pro- tection complex to elicit a very strong polarised block to replication (16). Here replication restart is inhibited by the Rtf2 protein, which promotes replication termination at RTS1 by the opposing fork merging with the stalled fork (17). However, some forks blocked at RTS1 can undergo recombination-dependent replication restart, and in so doing occasionally elicit genome rearrangements (5). Importantly most of this recombination in wild-type cells occurs independently of DSB formation, and there- fore presumably involves the unwinding of DNA strands at the stalled fork. To determine whether replication restart is the only way in which protein–DNA RFBs give rise to recombination we have investigated what the cellular response is to a persistent non-programmed bi-directional protein–DNA barrier. We show that, unlike RTS1, a persistent non- programmed barrier frequently results in DNA breakage even in the presence of a functional fork protection complex. However, unlike previously studied replication- associated DSBs, these breaks are not repaired by sister- chromatid recombination. Instead they are repaired by SSA and consequently are associated with DNA deletion. Intriguingly replication fork stalling and DNA breakage correlate with the appearance of structures that are analogous to ultraﬁne anaphase bridges (UFBs), which frequently form at fragile sites during mitosis in human cells, and are thought to occur when replication is incomplete (18,19). We propose that a failure of repli- cation restart at non-programmed bi-directional protein– DNA barriers results in a region of unreplicated DNA that becomes broken during mitosis or cytokinesis. Such breaks can only be repaired by allelic recombination, SSA or end-joining pathways due to the absence of an intact sister chromatid, demonstrating how some replication- associated DSBs can be predisposed to give rise to LOH and GCR. MATERIALS AND METHODS Media and genetic methods Media and genetic methods are based on established protocols (20). The complete and minimal media were Nucleic Acids Research, 2011, Vol. 39, No. 15 6569 yeast extract with supplements (YES) and Edinburgh minimal medium plus 3.7 mg/ml sodium glutamate (EMMG) plus appropriate amino acids (0.2475 mg/ml), respectively. Low adenine media (YES-LA) was supple- mented with 0.01 mg/ml adenine. Ade+ recombinants were selected on YES lacking adenine and supplemented with 200 mg/l guanine to prevent uptake of residual adenine (21). Where indicated 4 mM thiamine was added to media to repress expression from the nmt promoter. Strains, plasmids and probes The lacO array, family of repeats (FR) and MATa-con- taining strains were made by integration of derivatives of pFOX2 (22) (see below), which had been linearised with BlpI, at ade6-M375 in FO126 or FO1236. Southern blotting and/or colony PCR was used to determine that the linear plasmid had integrated at the correct site. The lacO array-containing derivative of pFOX2, pMW731, was constructed by subcloning a 4.5 kb XhoI lacO array fragment from pLAU43 (23) into the SalI site in pFOX2. Derivatives of pMW731 containing shorter lacO arrays were constructed by digestion and re-ligation to delete the appropriate number of repeats from the 115 repeat array. The digests were: NheI (to retain 2 lacO repeats in pMW892); NheI+BstEII (to retain 12 lacO repeats in pMW893); NheI+AfeI (to retain 15 lacO repeats in pMW894); and NheI+PshAI (to retain 21 lacO repeats in pMW895). The MATa cleavage site- containing derivative, pFOX16, was constructed by insert- ing a 117 bp EcoRV–HincII MATa fragment from pCla7 (24) into the HincII site that overlaps the SalI site in pFOX2. The derivative of pFOX2 containing 10 FR repeats (pMW897) was constructed by inserting a 838 bp XbaI–MluI fragment from AGP73 (25) into the SalI site in pFOX2. DNA ends were ﬁlled in using Klenow prior to ligation. pREP41/42/81-NLS-LacI-eCFP was constructed by PCR ampliﬁcation of lacI-eCFP from pLAU53 (23) using primers oMW708 (50-TTTGTCGACATGGGAAG TCCTAAGAAGAAACGAAAGGTGTTCACCGTGA AACCAGTAACGTTATACG-30) and oMW710 (50-TTT GGATCCTAATCTAGACACCATGG-30), which were designed to introduce a NLS and a SalI site at the 50-end of lacI-eCFP and a BamHI site at the 30-end. The ampliﬁed gene was then cloned into pREP41, pREP42 and pREP81 (26) using the SalI and BamHI sites. pREP41-NLS-EBNA1 was constructed by PCR ampliﬁ- cation of a portion of the EBNA1 gene encoding its DNA binding domain from plasmid 1160 (25) using primers oMW1367 (50-TATTACATATGGGAAGTCCTAAGA AGAAACGAAAGGTGTTCACCCGGGGTCAGGGT GATGGAGGCAG-30) and oMW1366 (50-TTGGATCCT CACTCCTGCCCTTCCTCAC-30). The ampliﬁed gene was then cloned into pREP41 using the NdeI and BamHI sites. pACYCREP81-HO has been described (24). All plasmids were sequenced to conﬁrm that no mu- tations had been introduced during the cloning. Probe A used for two-dimensional (2D) gel analysis in Figure 1 is a 3.7 kb BlpI–XbaI fragment from pMW731 and probe B is 3.1 kb SacII–BamHI fragment from pMW731. For the CHEF gel in Figure 4B probe I is a fragment spanning