Nucleic Acids Research, 2011, Vol. 39, No. 15 6573 Figure 2. Direct repeat recombination in strains containing a lacO-LacI array. (A) Schematic showing the ade6 direct repeat and lacO array on chromosome 3 and the two classes of Ade+ recombinant. Asterisks indicate the position of the point mutations in ade6-L469 and ade6-M375. (B) Neutral gel analysis of the XbaI lacO-containing fragment (see Figure 1A) from strain MCW1998 containing either pREP41 (LacI ) or pREP41-NLS-LacI-eCFP (LacI ++). Cells were grown for 20 h in the absence of thiamine prior to DNA extraction in agarose plugs. The Southern blot is probed with probe A (see Figure 1A). Lanes a and b, and c and d contain duplicate samples. (C) Ade+ recombinant frequencies in wild-type strains with and without the lacO array and LacI as indicated. (D) The percentage of Ade+ recombinants in C that are conversion-types. In all cases error bars are the standard deviations about the mean (see also Supplementary Table S1). percentage of stalled forks fail to restart in the presence of LacI (++) (Figure 1D and E). It is also important to note that the ascending Y-arc can contain small double Y structures, where both opposing forks are blocked and separated by at least half the total length of the DNA fragment being analysed. The same trends are seen with the lower level of LacI [LacI (+)], albeit they are far less marked than with the higher level (Figure 1C–E). To provide further evidence that replication forks stall within the lacO array we compared the changes in signal intensity along the Y-arc in a BamH1 fragment with those in an Xba1 fragment where the start of the lacO array is shifted 1 kb in from the fragment end (Figure 1G and H). In the BamH1 fragment a relative increase in replica- tion intermediates can be detected near to the start of the Y-arc (Figure 1G), whereas in the Xba1 fragment this increase is shifted along the Y-arc (Figure 1F and H), which is consistent with stalling occurring within the lacO array. Altogether these data indicate that the lacO- LacI interaction can act as a RFB in eukaryotes like in E. coli.