Replication Pausing and Imprinting analyzed fragment contains the abc region and the first 32 bp of the H1 domain. The plasmids were digested with MpsA1I and AfeI, which produce a 2.5-kb fragment for the empty vector. The membranes were probed with same fragment of the parental plasmid. Relative intensities of the pause signals observed in plasmids carrying the substitutions are given as percentages of the wild-type value below each 2D gel. (C) 2D-gel analysis of replication of plasmids with different DNA fragments inserted. doi:10.1371/journal.pgen.1001328.g007 observed when two plasmids containing P abc arrays were introduced (71% of wild-type sporulation and a 11.1% decrease in imprinting; Figure 8A, 8B). However, we were unable to detect a corresponding reduction in the intensity of the MPS1 signal, as a value of 11–12% is too small to be statistically significant when quantifying signals on 2D gels. Importantly, while these data indicate that a trans-acting factor(s) binds to the M and P abc regions, and is required for imprinting, sporulation and potentially pausing, they also show that out-titration of this factor is not the cause of the weaker pause signal observed in our plasmid assay compared to that at the chromosomal locus. We therefore considered whether chromatin structure of a locus might affect Figure 8. Out-competition of the putative trans-acting factor(s) that interacts with the M abc and P abc regions. (A) The effect of plasmids that carry arrays of the abc region on sporulation efficiency. Two different plasmids (constructed using pREP3X or pREP4X parental vectors) that each carries an array of approximately 10 copies of either the M abc or P abc region were introduced in the h90 wild-type background (strain JZ1), such that two plasmid-borne arrays of either M or P abc were present. The sporulation efficiency relative to that observed for a strain carrying empty vectors (100%) are given as a graph. The deviation is shown as error bars. (B) Quantification of mat1 imprinting in strains used in panel A. HindIII fragments were run and probed with the mat1P HindIII fragment. The intensity of the signal from the P abc array is higher due to greater sequence homology. Quantification of intensities of the DSB products relative to the strain that carries the empty vectors (100%) is given below. (C) Neither P abc nor M abc induces replication pausing at the transcriptionally silenced donor loci. Left, 2D-gel analyses of mat2P and mat3M replication are shown. Right, line drawings of the fragments analyzed are given. The direction of replication through each locus is indicated with grey arrows. The positions of the restriction enzyme sites and the probes used are shown. The sizes of the fragments are given. doi:10.1371/journal.pgen.1001328.g008 PLoS Genetics | www.plosgenetics.org 12 March 2011 | Volume 7 | Issue 3 | e1001328