contained the segment defined by Sub8, which is required for efficient pausing in our plasmid assay (Figure 7), therefore the reduction in imprinting is likely to result from the reduction in the replication pausing. Additionally, an inverted repeat in this region that flanks the site of the imprint may affect the imprinting process [18]. Therefore, the effects of a 12-bp substitution of a region located three bp centromere-proximal to the imprinting site, which substitutes one side of the inverted repeat present in the sequence, were investigated (Figure 9, HM-1). This substitution had no effect on the level of imprinting. Similarly, substitution of the 13-bp region located three bp centromere-distal to the imprinted nucleotides, which substitutes the other side of the inverted repeat, had no effect on imprinting (Figure 9, HM-2). Thus, the DNA sequence of neither the imprinted nucleotides nor the regions flanking them is of importance for the imprinting process. Discussion Replication barriers have been identified in the rDNA, at tRNA genes, and telomeric and centromeric sequences in several eukaryotic organisms, and at the RTS1 and MPS1 elements of the mating-type region in S. pombe (reviewed in [26]). The MPS1 site is particularly interesting as the replication pause leads to the introduction of the imprint marking the mat1 cassette in switchable cells of S. pombe, thereby establishing a functional link between DNA replication and cellular differentiation. Surprisingly, our analysis shows that cis-acting sequences required for MPS1 activity are located within the mating-type cassette DNA that is swapped during the switching process, such that the observed pause signal is constituted by forks stalled at not one, but two different sequences. Our results contrast a previously published study that showed that a 6-bp substitution, mut3, within the H1 domain flanking the mating-type cassette caused a significant reduction in imprinting and pausing [13]. This region is not required for pausing in our plasmid assay (Figure 7). Instead, by characterization of different deletions and substitutions, we were able to identify a 77-bp M- cassette region located 11 bp centromere-proximal to the site of the imprint that is required for both MPS1 activity and imprinting (Figure 6 and Figure 7). Importantly, a 2D-gel analysis of the corresponding region from the P cassette also detected a pause signal, despite the limited sequence identity between the two regions (Figure 7A, 7C). How these two cis-acting sequences induce replication pausing remains unknown, but most likely a trans-acting factor(s) mediates the activity (Figure 10, upper line drawing): Firstly, we have been unable to identify any secondary structure in the regions that could act as a barrier for the progressing replication fork. Secondly, MPS1 pausing is dependent on the trans-acting factors Swi1 and Swi3 that travel with the replication fork [24,35]. These factors are also required for replication pausing and termination at the S. pombe RTS1 element and at rDNA arrays where barrier activities are mediated by trans- acting factors [26]. Thirdly, we observed reduced mat1 imprinting and sporulation efficiency when plasmids containing arrays of M or P abc regions were introduced in otherwise wild-type background (Figure 8A, 8B). These data are consistent with the model that a trans-acting factor(s) required for imprinting, and by deduction replication pausing, is binding to the mat1 abc region and that out-titration occurs by competitive binding to the multiple plasmid copies present. Lastly, we were unable to detect any barrier activity at the donor loci mat2P and mat3M (Figure 8C). The K-region that separates the two donor loci has been shown to replicate early [33], suggesting that mat2P, but not mat3M, is replicated in the direction of replication where the barrier is active. Replication Pausing and Imprinting Also, this observation does not support the possibility that the secondary structure of the mat1 abc region mediates replication pausing, as the same structures would be expected to arise at the mat2P locus. Instead, it suggests that heterochromatin at the mat2P locus prevents the interaction of a trans-acting factor(s). The observation that the chromatin state of a cis-acting sequence affects its barrier activity is important as it establishes a mechanism, in addition to control of direction of replication, by which replication barriers could be regulated in other systems. Importantly, while we think a trans-acting factor(s) is required for pausing at both the mat1 cassettes, we are unable to predict, due to the limited similarity between the P abc and M abc sequences, whether it is the same factor(s) that is acting at the two sites. One interesting question is where the replication fork pauses in response to the MPS1 signal. Two lines of evidence suggest that the replication pausing takes place in the proximity of the imprint, and not further into the cassette as suggested earlier (Figure 10, arrow 1) [12]: i) We observe a leading-strand pause signal in the proximity of the imprint in the spacer deletion strain (SS13) where imprinting is abolished, but MPS1 pausing still occurs (Figure 5C). Importantly, it is not possible to distinguish a corresponding signal in the wild-type strain due to the leading-strand intermediates from stalled forks at the imprint that migrate to the same position [12,13]. ii) 2D-gel analysis detected a strong pause signal during the replication of a fragment containing only the imprint region and not the position where our previous analysis suggested MPS1 pausing occurred (Figure 5E). Instead, the weak leading-strand pause signal that we observed earlier in the vicinity of the strong priming site located approximately 350 bp into the cassette might reflect a minor pause of the replication fork while the priming event takes place. In this context, it is noteworthy that during bacteriophage replication, priming has been shown to pause leading-strand progression [36]. All the available data suggest that stalling of the replication fork at MPS1 is a prerequisite for imprint formation. However, the observation of pausing at MPS1 without subsequent imprinting indicates that pausing is only an initial step of the process by which the imprint is introduced. Our analysis identified a novel type of cis-acting element that is required for imprinting but not pausing. Deletion of a 205-bp region located on the centromere-proximal side of the pause signal abolished imprinting, but only slightly reduced pausing at MPS1 (Figure 2). Substitution of this region with a random sequence however, restored imprinting to 72% of wild-type levels, suggesting that this region mainly acts as a spacer to separate important flanking sequences. To our knowledge, this is the first description of such a genetic element. The important flanking sequences on one side of the spacer are defined by the abc region, while the flanking sequences of importance on the other side are unknown. However, the deletion of the 205-bp region leads to a complete abolishment of imprinting, therefore preventing us from using this assay to determine if the spacer extends further into the cassette. Also, we do not know whether extending the length of the spacer would affect imprinting; if this region is necessary for separation of two flanking sites, it is possible that enlarging it would prevent their concerted action. Alterna- tively, the wild-type spacer region could represent the minimum size and there will still be appropriate imprinting when it is lengthened. The reduction in imprinting caused by the spacer deletion appears not to be due to an inhibitory effect of transcription of the Mc gene, which is brought into close proximity of the imprinting site by the deletion, since neither repression of Mc transcription by growth in rich media nor deletion of the Mc promoter region leads to a restoration of imprinting (Figure 4). PLoS Genetics | www.plosgenetics.org 14 March 2011 | Volume 7 | Issue 3 | e1001328