6574 Nucleic Acids Research, 2011, Vol. 39, No. 15 Replication fork stalling at the lacO array correlates with an induction of recombination Having established that the lacO-LacI interaction hinders fork progression, we next tested whether this has any effect on recombination both between the repeats in the lacO array itself and the ade6 heteroalleles that ﬂank it (Figure 2A). In the case of the lacO repeats, LacI expres- sion results in an increase in contraction of the array as evident from the ladder of shorter lacO array-containing fragments seen on 1D gels (Figure 2B). In the case of the ade6 repeats, recombination was measured by determin- ing the frequency of Ade+ recombinants (Figure 2A and C). Here recombinant formation increases by 15-fold with LacI (+) and almost 700-fold with LacI (++) (Figure 2C and Supplementary Table S1). Indeed with LacI (++) the frequency of Ade+ recombinants appears to be at or near its maximum with the remaining cells either being Ade deletion-types or having lost the lacO array or LacI ex- pression (data not shown). Heightened recombination is dependent on the presence of both the lacO array and the expression of LacI that is able to localize to the nucleus by the inclusion of a NLS (Figure 2C and Supplementary Table S1). The presence of a his3+ gene between the ade6 repeats enables us to distinguish two types of Ade+ recombinant—those that retain his3+ (conversion-types) and those that lose it (deletion-types). In the absence of LacI 78% of spontaneous recombinants are deletion- types (Figure 2D and Supplementary Table S1). Strikingly this percentage increases signiﬁcantly with expression of LacI reaching 99% of recombinants with LacI (++) (Figure 2D and Supplementary Table S1). Altogether these data suggest that replication fork stalling at the lacO-LacI RFB triggers recombination, which mostly results in dele- tions. Intriguingly, this contrasts with recombination induced by the RTS1 RFB, which yields approximately equal amounts of deletion- and conversion-types (14), and sug- gests that programmed and accidental protein–DNA RFBs might trigger recombination in different ways. The genetic requirements of lacO-LacI-induced recombination indicate that deletions arise by SSA We have previously shown that the majority of recombin- ants that are induced by the RTS1 RFB (both conversion- and deletion-types) are dependent on Rad51 (14). To see if the same is true with the lacO-LacI RFB we assessed Ade+ recombinant formation with different levels of LacI ex- pression in a rad51D background (Figure 3A and C and Supplementary Table S1). In all cases loss of Rad51 has Figure 3. Genetic requirements for lacO-LacI-induced direct repeat recombination. (A) Ade+ recombinant frequencies in lacO array-containing strains expressing LacI (+). (B) The percentage of Ade+ recombinants in A that are conversion-types. (C) Ade+ recombinant frequencies in lacO array-containing strains expressing LacI (++). (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 and Supplementary Figure S1).