6582 Nucleic Acids Research, 2011, Vol. 39, No. 15 acts as a bi-directional RFB we believe that UFBs are most likely caused by a failure to complete replication in this case. This problem is far less likely with programmed barriers such as RTS1, which are unidirectional thereby allowing the completion of replication by the opposing fork if restart fails. In human cells it is thought that entangled sister chro- matids can be broken during anaphase by the pulling forces exerted by the spindle as there are generally enough microtubules (>10) attached to each kinetochore to do this (50). However, in S. pombe each kinetochore is contacted by only 2–4 microtubules and therefore the forces applied to each chromosome are insufﬁcient to rupture them during anaphase (51). Indeed in S. cerevisiae the breakage of sister chromatids that remain catenated due to topoisomerase II inactivation depends on cytokin- esis (52). In our study we observe UFBs that persist up to the point that the cell division septum is formed suggesting that DNA breaks occur during cytokinesis. Protein–DNA arrays magnify a problem that is normally efﬁciently dealt with by the cell Replication forks have to negotiate a multitude of protein–DNA interactions whilst replicating the genome. The machinery that enables them to do this includes fork protection factors such as Swi1 and Swi3 (= Tof1 and Csm3 in S. cerevisiae), the putative sweepase activity of Pfh1 (= Rrm3 in S. cerevisiae), and HR proteins that promote replication restart when replisome disassembly has occurred (5,39,40,53,54). Disabling any of this ma- chinery can result in altered pausing at protein–DNA barriers and elevated levels of genome instability (5,15,40,55). A prime example of this is the pausing of rep- lication forks at tRNA genes, which only elicits a strong recombinational response in the absence of fork protec- tion or barrier removal activities (15,56). Presumably even when it is fully functional it occasionally fails to cope with the barriers that it is presented with. By using protein– DNA arrays we believe that we have increased the fre- quency of these normally rare, but nonetheless signiﬁ- cant, events by creating a particularly potent barrier. Interestingly array size does not appear to be the most im- portant factor in determining the potency of the barrier, as similar levels of anaphase bridge formation and recombin- ation are obtained with a 12 repeat lacO array as with a 115 repeat array, and even two repeats produce measur- able effects. More important than the number of repeats appears to be the amount of protein that is available to bind to them. Wild-type cells appear to cope well with a low-level of LacI-eCFP [non-induced LacI (+) level] that is sufﬁcient to produce a detectable focus of ﬂuroscence at the 115 repeat lacO array, however increasing the level of LacI-eCFP by 10-fold [LacI (+) level], which still pro- duces just a single focus of ﬂuorescence in cells with the array, produces signiﬁcant levels of genome instability that are further increased when factors such as Pfh1 are missing. A further 10-fold increase in LacI-eCFP [LacI (++) level] is sufﬁcient to ﬁll the nucleus such that a discrete focus of ﬂuorescence is no longer detectable, and at this level 15–20% of cells exhibit a UFB and DSB in each cell cycle. Presumably high concentrations of LacI make passage through the barrier difﬁcult because displaced protein is more rapidly replaced. Nevertheless, even with LacI (++) the majority of cells appear to suc- cessfully replicate the lacO array in each cell cycle indi- cating that fork protection and replication restart are reasonably efﬁcient even when faced with a particularly challenging barrier. The cell’s last ditch effort to restart replication? It has recently been shown that Rad22 and, to a lesser extent, Rad51 are needed for promoting replication restart at the RTS1 RFB (5). In our study we observe increased levels of UFB formation when Rad22 and Rad51 are absent consistent with them promoting replication restart at the lacO-LacI RFB. We also observe a lacO-LacI- dependent induction of Rad51-dependent conversion- type recombinants that might be generated during attempts to restart replication. Moreover, in those cells where lacO-LacI induces problems in chromosome segre- gation we observe Rad22-YFP foci that persist through- out mitosis suggesting that attempts to restart replication continue right up to the point of cytokinesis. As men- tioned earlier this is reminiscent of the situation in human cells where DNA repair proteins such as FANCD2 and BLM are present at replication intermedi- ates that persist into anaphase (18,19). Even though there is a cellular response mounted to the replication problem this does not appear to include an obvious delay in cell cycle progression, since a signiﬁcant percentage of cells displaying UFBs divide at a normal division cell length (data not shown). We suspect that cells dividing with UFBs at greater than normal division cell length do so as a consequence of DNA breakage caused by replication fork stalling in the previous cell cycle. As DSB repair can occur by annealing between lacO repeats, cycles of fork stalling and DNA breakage can occur in successive cell cycles until the array is com- pletely deleted. So if cell elongation is solely due to DNA breakage then replication fork blockage at the lacO array and presumed under replication does not appear to strongly induce a checkpoint response. An absence of checkpoint activation by forks arrested at lacO-LacI would be similar to what has been observed at both the RTS1 and Fob1-mediated RFBs (6,54). There are also mutants that fail to complete replication and progress to anaphase without inducing a checkpoint response even though the checkpoint machinery is not obviously disabled (57,58). It is therefore reasonable to suppose that unrepli- cated DNA at the lacO array could go unnoticed by the checkpoint machinery. EBNA1 and genome instability EBV is associated with a variety of human malignancies (42), and this association appears to depend on the expres- sion of EBNA1, which is known to induce chromosomal aberrations and DNA DSBs in malignant B cells (59). Such genome instability is thought to result from incr- eased levels of DNA damage caused by transcriptional activation of NOX2 (60). However, our data suggest