Replication Pausing and Imprinting analysis are marked. The H1 domain is represented as a grey rectangle. The imprint is shown as a black circle. The positions of the restriction enzyme sites used are indicated by vertical lines. (E) 2D-gel analysis of the mat1P and mat1M imprint regions contained in DraI fragments. Migration of size markers after the first-dimension electrophoresis step is shown below each 2D gel. A line drawing of the region is given on right. The positions of the mapped priming site and the probes are shown. The site of the imprint is indicated with a circle. doi:10.1371/journal.pgen.1001328.g005 affects where primers are put down during lagging-strand replication of the region. Surprisingly, a signal in the proximity of the imprinting site was detected in this strain (Figure 5B). This signal is not due to hydrolysis of imprinted DNA, as we did not observe a signal where the centromere-proximal fragment would migrate. Instead, it suggests that a priming event is taking place at the site of imprinting, although this was undetected by our RIP mapping analysis [12]. To test if we could also observe leading- strand pausing at this position, we analyzed the leading-strand replication intermediates from the same strains (Figure 5C). A signal was detected at the site of the imprint in both the wild-type strain and strain SS25, which corresponds to previously observed stalling of leading-strand replication one nucleotide before the imprint [12,13]. However, in the strain carrying the spacer deletion (SS13) and therefore lacking the imprint, a weaker signal can also be observed at this position. This suggests that leading- strand replication pausing at the MPS1 site occurs in the proximity of the imprinting site. These results makes us reconsider our data presented earlier where we had mapped the paused replication fork at MPS1 to a position approximately 350 bp centromere-proximal to the imprinting site [12]. In order to discriminate between these two models, we performed a 2D-gel analysis of a small DraI fragment, which included the region of imprinting but not the region where we had previously proposed that stalling of the replication fork occurred (Figure 5E, left panel). The presence or absence of the pause signal on the Y-arc would determine which model is correct. The analysis clearly detected a strong pause site at a position that corresponds well to the site of the imprint. A similar analysis of the P cassette gave the same result (Figure 5E, right panel). Thus, the data suggest that the MPS1 signal stalls the replication fork at the region of imprinting and not within the cassette. Definition of the MPS1 signal sequences To define the sequences required for the MPS1 signal, we analyzed the 111-bp region deleted in the strain SS2 (Figure 2) by dividing it into three shorter segments, named a, b and c (Figure 6A). Deletion of each segment individually led to a complete abolishment of imprinting and a dramatic reduction in replication pausing (10%, 9% and 23% residual pausing when region a, b and c was deleted, respectively; Figure 6). Substitution of regions a and b with random sequences had similar effects to deleting them, resulting in abolished imprinting and reduced replication pausing to about 10% of wild-type levels (Figure 6). This demonstrates that these sequences are required for pausing and imprinting. Substitution of region c, which almost entirely consists of thymidines, with a random sequence caused only a reduction in imprinting to 28.4% of wild-type levels and a 47% reduction in pausing (Figure 6). Therefore, although this region clearly plays a role in both imprinting and pausing processes, there is not an absolute requirement for a specific sequence. The observation that deletion of region c abolishes imprinting, whilst replacement of it leads to only a reduction suggests that region c partly acts as a spacer. To investigate if the abc region could act as a replication barrier when transposed to a different locus and to define the sequences sufficient for barrier activity, we inserted the region into the plasmid pREP81K, next to the origin of replication [31]. Initially the transposed sequences included the abc region and the first 32 nucleotides of the H1 domain (Figure 7B, line drawing), as an earlier study had showed that a six-bp substitution within this region, called mut3, significantly reduced replication pausing [13]. Also, the first 32 nucleotides is what is left of the H1 domain in the strain carrying the cis-acting 263-bp smt0 deletion that abolishes imprinting, but does not affect replication pausing at MPS1 (see Introduction). The experiments were performed in a genetic background where the mat1 locus and the mat2P and mat3M donor loci had been deleted to avoid interference due to sequence homology (strain SS61). 2D-gel analysis of the plasmid detected replication pausing at a position on the Y-arc that corresponded to the position of the inserted sequences, whilst no barrier activity could be observed for the empty plasmid (Figure 7B, left panels). This plasmid-based system was used to further define which sequences within the abc region were required for replication pausing. Ten 11–12 bp substitutions were introduced, covering the entire abc region (Figure 7A). The substitutions Sub1, Sub2, Sub9 and Sub10 had little effect on pausing, however, the six substitutions within the middle of the abc region all reduced pausing to a significant degree (Figure 7B). Sub4, Sub5, Sub6 and Sub8 displayed about 20% residual pausing, while slightly more pausing could be observed for Sub3 and Sub7 plasmids (39% and 49% of the wild-type, respectively). Efficient pausing at MPS1 therefore requires at least the middle 66 bp of the abc region. The abc region alone and two shorter fragments comprising of the regions covered by the Sub4 to Sub8 and Sub4 to Sub9 substitutions (M abc, M R48 and M R49, respectively) were analyzed to determine if they were sufficient for pausing without the additional sequences of the H1 domain (Figure 7C; left panels). Omitting the H1 sequences had no effect on pausing, indicating that the region defined by the mut3 substitution is not important in this system. Significant pause signals could be observed for both the Sub4 to Sub8 and Sub4 to Sub9 fragments, the latter being of a similar intensity to the full abc region. Thus, the data suggest that the 77- bp Sub3-Sub8 region located 11 bp centromere-proximal to the imprinted nucleotides at mat1 is required and sufficient for maximum replication pausing in this assay. This requirement for specific sequences within the mat1M cassette was very surprising because replication pausing is observed both when mat1 contains P- and M-cassette DNA, which generally share no sequence similarity. We therefore analyzed the sequence corresponding to the M abc region in the P cassette, to see if replication pausing was also induced by this fragment. Indeed, a strong pause site could be observed by 2D-gel analysis (Figure 7C, P abc). Interestingly, an alignment of the two regions shows that there is a low level of sequence similarity between these M- and P-cassette segments, which is greatest in the region covered by substitutions Sub4 to Sub9 (Figure 7A). However, 2D-gel analysis of shorter segments of the P cassette, which correspond to the Sub4 to Sub8 and Sub4 to Sub9 regions of M abc, did not detect any pause signals (Figure 7C, P R48 & P R49). Thus, while there is low similarity between the P and M abc sequences, the size of the region required for pausing differs. Does a trans-acting factor(s) interact with the abc region? The replication pause signal observed in our plasmid assay is not as intense as that at the chromosomal mat1 locus (compare WT PLoS Genetics | www.plosgenetics.org 9 March 2011 | Volume 7 | Issue 3 | e1001328