6572 Nucleic Acids Research, 2011, Vol. 39, No. 15 Table 1. Schizosaccharomyces pombe strains used in this study Mating type Genotype Strain FO126 MCW1221 MCW429 FO1236 MCW1988 MCW2087 MCW2088 MCW2089 MCW3350 MCW3409 MCW2189 MCW2186 MCW4087 MCW2372 FO1751 MCW2669 MCW4916 MCW4949 SAS67 MCW5157 MCW5158 MCW5159 MCW5160 MCW5162 MCW5320 h h+ h+ h h h+ h+ h+ h h+ h+ h h+ h+ h h+ h+ h+ h+ h+ h+ h+ h+ h+ h ura4-D18 leu1-32 his3-D1 ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 rad51D::arg3+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 rad22D::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 rad22D::ura4+ rad51D::arg3+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ ade6-M375 swi10D::kanMX6 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 swi10D::kanMX6 rad22D::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ ade6-M375 rqh1D::kanMX6 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 exo1D::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 rqh1D::kanMX6 exo1D::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ ade6-M375 rad50S ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 rad22+-YFP-kanMX6 ura4-D18 leu1-32 his3-D1 arg3-D4 rad22+-YFP-kanMX6 ura4-D18 leu1-32 his3-D1 arg3-D4 L469/pUC8/his3+/lacO115/ade6-M375 leu1-32::pJK148-leu1-pfh1+ pfh1D::kanMX6 ura4-D18 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ ade6-M375 leu1-32::pJK148-leu1-pfh1-mt* pfh1D::kanMX6 ura4-D18 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/ lacO115/ade6-M375 chk1D::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO2/ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO12/ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO15/ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO21/ade6-M375 ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/FR10/ade6-M375 hht2+::GFP-ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4 ade6-L469/pUC8/his3+/lacO115/ade6-M375 Source Lab strain Lab strain Lab strain Lab strain This study This study This study This study This study This study This study This study This study This study This studya This study This studyb This studyc This study This study This study This study This study This study This study aDerivative of SP220 (63). bDerivative of ySP293 (41). cDerivative of ySP377 (41). recombination in S. pombe (14). As RTS1 is a progra- mmed protein–DNA barrier we wanted to see whether a non-programmed protein–DNA barrier provokes a similar response. In Escherichia coli both lac and tet operator arrays when bound by their cognate repressor can act as site-speciﬁc protein–DNA RFBs (31). To deter- mine whether the same is true in eukaryotes we inserted 115 lacO repeats between a direct repeat of mutant ade6- heteroalleles on chromosome 3 of S. pombe in approxi- mately the same position that RTS1 had been placed pre- viously (Figure 1A) (14). The Lac repressor protein (LacI), with an N-terminal nuclear localization signal (NLS) and C-terminal enhanced cyan ﬂuorescent protein (eCFP) tag, was then expressed from the thiamine-repressible nmt promoter in either pREP81 or pREP41. The nmt pro- moters in these vectors are differently mutated resulting in an 10-fold difference in expression levels, which is evident from the amount of ﬂuorescence in cells following induction (Figure 1B). pREP81 levels of LacI [hereafter referred to as LacI (+)] result in a discrete focus of ﬂuor- escence due to a high proportion of the induced protein binding to the lacO array, whereas pREP41 levels [here- after referred to as LacI (++)] ﬁll the entire nucleus (Figure 1B). Induction from the nmt promoter following the removal of thiamine is quite slow with the ﬁrst mRNA detectable after 10 h and maximal protein expression ach- ieved after 16 h (32). In our hands the maximum level of LacI is achieved after 20 h (data not shown). Much of our analyses therefore focus on samples taken after 20 or 24 h, with the latter time point ensuring that most cells have completed at least one cell cycle with LacI at its maximum level. After 20 h of LacI induction, replication across the BamHI DNA fragment ( 5.2 kb) containing the lacO array was analysed by native 2D gel electrophoresis (Figure 1A and C). In both the presence and absence of LacI an arc of Y-shaped replication intermediates is detected. However, LacI (++) results in a greater accumu- lation of replication intermediates in the ascending portion of the Y-arc than when it is absent (Figure 1D). Replication across the ade6 locus is essentially unidirec- tional [moving towards the centromere (cen3)] (Figure 1A) (14), and therefore the accumulation of replication inter- mediates in the ascending Y-arc indicates that the progres- sion of forks is perturbed as they enter the array. The absence of a deﬁned point of DNA accumulation on the ascending Y-arc indicates that forks stall at multiple sites within the array. There is also a corresponding decrease in the descending Y-arc signal together with an increase in converged forks indicating that a signiﬁcant