Reduced imprinting is also not due to a repressive effect of the strong priming site that we have mapped previously, which is located in the region flanking the spacer on the centromere- proximal side, as extension of the deleted region to include these sequences does not restore imprinting (Figure 4E). This priming site is also not directly required for imprinting or pausing, as deletion of it only causes minor reductions in the efficiencies of these processes (Figure 2). An alternative explanation is that it is not the specific sequence, but the chromatin structure of the transcribed Mc region that inhibits imprinting in the absence of the spacer. One possibility is that there is a requirement for a non-transcribed spacer, as for example the nucleosome arrangement in such a region might affect where primers are synthesized during lagging-strand replication. Using high-resolution Southern analysis of replication intermediates, we attempted to map the priming sites used in the region of the wild-type, spacer-deleted and spacer-substituted strains. In the imprinting-proficient wild-type and spacer- substituted stains, we detected a putative priming signal within the mating-type cassette, approximately 350 bp from the site of the imprint, at a position that corresponds to where we earlier detected a strong priming site using the RIP mapping method (Figure 5B) [12]. Another putative priming signal was observed approximately 30 bp centromere-proximal from the imprinting site, which was undetected by our earlier RIP mapping analysis. Both these signals were absent in replication intermediates from the strain carrying the spacer deletion, suggesting that the non- transcribed spacer region indeed influences the sites of primer synthesis after the replication fork is released from pausing at MPS1 (Figure 10, arrows 3 & 7). If the imprint is introduced by ligation of a not fully processed Okazaki fragment to the subsequent fragment, the length and position of these could be crucial, maybe due to topological constrains during the replication process. Interestingly, a putative priming signal is detected at the site of imprinting in the spacer deletion strain (Figure 5B and Figure 10, arrow 2). Because of the strong signals created by hydrolysis of the imprinted DNA, it is impossible to determine whether such a priming event also occurs at this position in the wild-type strain. This priming site was not observed for the wild-type strain in our previous RIP mapping analysis [12]. However, it is possible that priming at this site is special, involving a shorter RNA primer that does not protect the fragment from l-exonuclease digestion used for RIP mapping [29]. Also, the high-resolution Southern analysis is a more direct method for analysis of replication intermediates than RIP mapping. The detection of specific priming at the imprinting site does provide strong support for a model proposing that the imprint is introduced by a mechanism involving the fusion of a not fully processed Okazaki fragment to the subsequent fragment (Figure 10, arrow 4). In support of this possibility, DNA polymerase alpha (Swi7), which initiates lagging-strand replication together with primase, is involved in imprinting [9]. Another observation that supports a model for the imprinting process that involves a priming event is that we were not able to detect any sequence requirements for either the two imprinted nucleotides or the 12-bp and 13-bp regions flanking the site of the imprint (Figure 9). This observation is consistent with another study, in which substitution of five of the first six bp of H1, including one of the imprinted nucleotides, as well as the 9th to the 14th bp, had no effect on imprinting levels [13]. Similarly, we observed that whilst deletion of the last 34 bp of the M cassette that flank the imprinted nucleotides (region c) abolished imprint- ing, substitution of this region only caused a reduction in imprinting levels (Figure 6). Therefore there appears to be no Replication Pausing and Imprinting absolute sequence requirement at or around the site of the imprint for the imprinting process to take place. Additionally, region c may partially act as a spacer element, again highlighting the potential importance of the position of priming events in the imprinting process. Thus, our data suggest that the imprint is introduced at a certain distance from the replication pause site in a sequence- independent manner. The position of this priming event might not only affect whether the imprint is formed, but also whether it is maintained. Here, the distance to the sequences within the H1 domain, which has already been implicated in the maintenance of the imprint, might be important (Figure 10, arrow 5) [13]. Our data presented here suggest that ribonucleotide imprints might be introduced at other replication barriers, but not maintained. Such temporary marks could be important for replication-associated processes such as DNA damage repair or the establishment of certain chromatin. Also, the production of epigenetic marks during the replication process and subsequent asymmetric segregation of the differentiated sister chromatids may be a more general mechanism for cellular differentiation and development in eukaryotes. Understanding the process of imprinting in fission yeast and identifying the cis- and trans-acting factors involved will help to establish whether this specific type of epigenetic mark also plays a role in cellular differentiation of higher eukaryotes. Finally, unravelling the molecular processes at the replication fork barriers involved in imprinting may also increase our understanding of the events that occur at uninten- tional replication barriers and how they lead to genome instability. Materials and Methods Construction of S. pombe strains Deletions and substitutions were introduced into the M cassette using inverse PCR on plasmids containing mat1M DNA. Deletions and substitutions were verified by sequencing. The mutated fragments were subsequently transformed into the strain EG571, which carries the ura4+ gene inserted at the H1 domain and is deleted for the donor loci mat2P and mat3M (gift from Olaf Nielsen). 5-fluoroorotic acid-resistant colonies were selected, and the replacement of the parental ura4+-containing allele of mat1 with the mutated allele was verified by PCR, followed by sequencing and Southern analysis. The sequences of the substituted segments were: Strain SS25, AATTGAACGTTGTTTTTCCATTTTG- TATAAGCCGTCGCCGGTT TAGAAGCGTTGAATGTCAG- GTATTATGAATAGTAAAATCTTATTTATCTATTTTGAA- GGTCAATTGTTATCATTTCTTAAAGTGTGATTGTTATA- TATCAGTAGGTTTAAATATATTAACACTAGATAAATTCT- TGTCTCATTATTTATTGATTCCACATAA; Sub a, CTGTG- TCTGTCGAGTTGCCCATCAGTACGGAGCTAAAACGAA- T; Sub b, GTAA-GTCACCGATAAACGCGCCTGGAATGTT- TCT; Sub c, TTTGATCGCCTGGA-ACCATGAACGACCT- AAGGTC; Sub 1-10, TCTCTGCAGGCT, CACTGCAGCGT, CGCTGCAGATC, TTCTGCAGGCC, CCCTGCAGGAA, GT- CTGCAGGTA, TTCTGCAGCTC, TCCTGCAGGTC, GG- CTGCAGCGC, GCCTGCAGGAT, respectively. The recipes of media and the method for the transformation procedure are given in [37]. Quantification of imprinting levels by Southern blot analysis Unless otherwise stated, the chromosomal DNA was purified from cells grown in PMA+ media as described in [10]. The DNA samples were digested with the HindIII restriction enzyme and separated on a 0.6% agarose gel. Southern analysis was performed with a probe specific to the 10.6-kilo base pairs (kb) mat1P HindIII PLoS Genetics | www.plosgenetics.org 16 March 2011 | Volume 7 | Issue 3 | e1001328