Replication Pausing and Imprinting Figure 6. Characterization of a cis-acting region named abc that is required for replication pausing and imprinting. (A) Left, line drawing representation of the strains used. A gap represents a deletion and a gray line segment represents a substitution of a DNA sequence. The defined abc region is shown and the borders of the element are indicated by vertical lines. Right, names of the strains and quantification of DSB products and pause signals on 2D gels (including standard error) are given. (B) Southern analysis of the strains shown in panel A. See Figure 2 legend for method. (C) 2D-gel analysis of the strains shown in panel A. See Figure 2 legend for method. doi:10.1371/journal.pgen.1001328.g006 signals in Figure 2C and Figure 7B). This is partly due to the fact that even though the abc region is inserted next to the origin in the plasmid, it is replicated in both directions [32], and that only the forks moving in the same direction as at the genomic mat1 locus give rise to a barrier signal. However, we think that the weaker signal cannot be fully explained by this observation. Another possibility was that sequences outside the abc region contributed to pausing in the chromosomal context. However, inclusion of a 323- bp sequence centromere-proximal to abc or the entire centromere- distal H1 domain did not increase the intensity of the pause signal in our plasmid assay (data not shown). Thus, flanking sequences are not required for more efficient pausing. Alternatively, the plasmid used is present in multiple copies and therefore there could be an out-titration of a putative abc-interacting trans-acting factor(s) required for pausing. To test this possibility, we introduced plasmids containing the M abc or P abc region into h90 wild-type background to examine if an inhibitory effect could be observed on imprinting and switching, which both depend on MPS1 pausing. No effect was observed. We then constructed plasmids with arrays of the M or P abc region to increase the copy number. Each array contained approximately 10 tandem copies. Introduction of two different plasmids containing M abc arrays led to a reduction in sporulation to 57% of the wild-type levels and a 11.8% decrease in imprinting (Figure 8A, 8B). A similar effect was PLoS Genetics | www.plosgenetics.org 10 March 2011 | Volume 7 | Issue 3 | e1001328