mus81 and probe II is a fragment spanning brl1. The probe for the CHEF gel in Figure 4D is a 1 kb fragment amp- liﬁed from genomic DNA using primers oMW1355 (50-GC GCAACTCCTTTAGGGGTAG-30) and oMW1356 (50-G CGTATTCTAACGTCTTCGTG-30). A full list of S. pombe strains used in this study is given in Table 1. Recombination assays Spontaneous, lacO-LacI-induced and FR-EBNA1-induced Ade+ recombinant frequencies were measured essentially as described (14,21). To select for plasmids and allow for expression of LacI/EBNA1, cells were grown on EMMG lacking leucine and thiamine for 6–7 days at 30 C prior to selecting Ade+ recombinants. Recombinant frequencies represent the mean value from at least 15 colonies for each strain, and for strains con- taining plasmids at least three independent transformants were tested. The statistical signiﬁcance of differences between recombinant frequencies was calculated with two-sample t-tests. Viability assay Strains with and without the 115 repeat lacO array con- taining either pREP41 or pREP41-NLS-LacI-eCFP were grown on EMMG plates lacking histidine and leucine and containing thiamine. Colonies from these plates were used to inoculate EMMG liquid media lacking histidine, leucine and thiamine, and these cultures were grown for 24 h before cells were harvested, washed in sterile water, counted and plated onto YES-LA containing thiamine. Plates were incubated at 30 C for 3–5 days before colonies were counted to determine the percentage of the plated cells that were viable. In addition colonies were scored for whether they were Ade+ (white/pale pink) or Ade (dark pink/red) to provide a measure of the percent- age of viable cells that were recombinants. S. pombe genomic DNA preparation Genomic DNA for neutral/neutral 2D gels was puriﬁed from logarithmically growing cultures as described (27), and enriched for replication intermediates on benzoylated naphthoylated DEAE (BND)-cellulose (Sigma). Genomic DNA for standard 1D agarose gels and CHEF gels was prepared in agarose plugs either as described by Win et al. (28) (Figures 2B and 4B) or as described by Hyppa and Smith (29) (Figure 4D). For the data in Figure 4D the plugs were melted at 65 C for 5 min prior to digestion of the agarose with ß-agarase I for 2.5 h at 37 C. The DNA was then digested over night with 40 units of AvrII. For the data in Figure 2B the DNA was digested in the plugs by overnight incubation with 40–100 U of restriction enzyme at 37 C. Nucleic Acids Research, 2011, Vol. 39, No. 15 6571 Gel electrophoresis Two-dimensional gels were run according to Brewer and Fangman (30). Agarose gels of 0.4 and 1% were run for the ﬁrst and second dimension, respectively. Neutral 1D gels were 0.8% agarose and were run in 1 TBE at 50– 60 V for 16 h. The CHEF gels in Figure 4B were 0.7% agarose in 1 TAE, and were run for 48 h at 2 V/cm with an initial switch time of 20 min and a ﬁnal switch time of 30 min using a CHEF II apparatus (Bio-Rad). The CHEF gel in Figure 4D was 1.0% agarose in 0.5 TBE and was run for 17 h at 6 V/cm with a switch time of 1–6 s using a CHEF II apparatus (Bio-Rad). All gels were Southern blotted and probed with 32P-labelled probe as indicated. Analysis of the blots was performed using a Fuji FLA3000 PhosphoImager and Image Gauge software. Microscopy For imaging UFBs, cells were grown in EMMG lacking leucine and thiamine for 24 h at 30 C to allow for NLS-LacI-eCFP expression. Cells were then ﬁxed in 70% ethanol, stained with DAPI or SYBR Green and analysed using an Olympus BX50 epiﬂuorescence micro- scope equipped with the appropriate ﬁlter sets to detect blue and green ﬂuorescence. Black and white pictures of the single ﬂuorescence channels were acquired with a cooled CCD camera (Princeton Instruments Inc., NJ, USA) controlled by Metamorph software (version 6.1r6; Molecular Devices, Sunnyvale, CA, USA). Single channel pictures were assigned pseudocolours and merged using Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA). Live cells were imaged on an inverted Olympus IX71 microscope controlled by a DeltaVision Core using DeltaVision softWoRx 4.0.0 software (Applied Precision Inc., Issaquah, WA, USA), all subse- quent image manipulations were performed with this software suite as well. Images were taken with a CCD camera (CoolSNAP HQ; Photometrics, Tucson, AZ, USA) using an oil-immersed Olympus 100 UPlanSApo objective with a of 1.40. For time-lapse analysis cells were mounted with soybean lectin (Sigma-Aldrich Ltd., Dorset, UK) in a glass bottom culture dish (MatTek Corp., Ashland, MA, USA) and observed in the appro- priate medium at 30 C. A stack of 26 focal planes at a step-size of 0.2 mm was taken every 5 min for 4 h. RESULTS A lacO array bound by LacI can perturb replication fork progression in eukaryotes We have previously shown that replication fork stalling at the polar RFB RTS1 induces Rad51-dependent Figure 1. Continued amount of LacI. Note that the shadow behind the Y-arc is due to shearing of DNA during sample preparation, and, in the case of LacI (++), also contraction of the lacO array (see also Figure 2B). (D and E) Quantiﬁcation of data like in (C). The data are mean values from three independent experiments. Error bars represent standard deviations. (F) Two-dimensional gel analysis of replication intermediates in the XbaI lacO-containing fragment from a wild-type strain with the indicated amount of LacI. (G and H) Quantiﬁcation of data like in (C) and (F). The diagram inset in (G) shows the regions of the Y-arc that were quantiﬁed. The data are mean values from three or four independent experiments. Error bars represent standard deviations.