Author Summary Differentiated sister chromatids, coupled with non-random segregation, have been proposed to control cell fate during differentiation in multicellular organisms as well as fission yeast. However, while nothing is known about how the differentiated sister chromatids are established in higher eukaryotes, the nature of the epigenetic mark that is required for the asymmetrical switching pattern of Schizosaccharomyces pombe is known. We have previously shown that two ribonucleotides are introduced in a strand- and site-specific manner during DNA replication at the mat1 locus in only one of the two sister chromatids synthesized. However, the molecular mechanism by which the imprint is introduced is elusive. We know that imprinting involves a site-specific pause of the replication fork, but how the replication fork is paused and how this leads to imprinting were unknown. Here we present key novel discoveries important for unravelling this mecha- nism. Our data suggest that site-specific replication pausing leads to lagging-strand priming at the site of imprinting and that this RNA primer is converted into the imprint by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. Potentially, this could be a more general mechanism for formation of epigenetic marks. the intact DNA, and have shown that subsequent hydrolysis of the imprint can be achieved using RNAse T2 or alkali treatment [12]. In addition, we have shown that when DNA from a wild-type strain is purified under conditions that are known to hydrolyze RNA, a one-nucleotide gap can be detected in the imprinted strand [17]. This study also detected the presence of a 39-terminal ribonucleotide, suggesting that the wild-type imprint consists of two ribonucleotides. A separate study using a mutant strain where a PstI restriction site has been introduced at the site of the imprint detected a nick with a 59 hydroxyl at the site of the imprint [20]. Importantly, the presence of a 59 hydroxyl is expected from the hydrolysis of a DNA-RNA-DNA hybrid molecule. Several trans-acting factors have been identified that are required for the introduction of the imprint [21]. These include the checkpoint proteins Swi1 and Swi3 that travel with the replication fork and DNA polymerase alpha (Swi7), which is predominantly involved in lagging-strand replication [9,22,23]. Imprinting also correlates with a replication pause occurring at the site named MPS1, which can be observed both in P and M cells in the proximity of the imprint and is dependent on the trans-acting factors Swi1 and Swi3, but not on Swi7 [12,24]. Replication pausing and imprinting are dependent on the direction of replication at the mat1 locus, which is tightly regulated by a site- specific polar replication termination site named RTS1 [25]. RTS1 is located at the centromere-proximal side of mat1 (Figure 1B), such that mat1 is only replicated by forks originating from the centomere-distal direction. Changing this direction of replication through mat1, by introducing replication origins on the centro- mere-proximal side, by inverting the mat1 locus relative to the RTS1 element, or by transposing the RTS1 element to the centromere-distal side of mat1 in the inverted orientation, leads to a reduction or an abolishment of imprinting [10,25]. Inversion of the mat1 locus relative to the RTS1 element also leads to abolishment of replication pausing at MPS1, showing that the signal for pausing only acts on replication forks moving in one direction [24]. Such polarity is observed for all known site-specific replication barriers (reviewed in [26]). Replication Pausing and Imprinting Various cis-acting sequences involved in pausing at MPS1 and imprinting have been identified. These include the smt0 deletion that removes 263 base pairs (bp) starting at a position 33 bp centromere-distal to the mat1 cassette within the H1 homology domain and extending distally [27]. The smt0 mutation leads to the abolishment of imprinting but does not affect pausing at MPS1 [24,27]. A smaller deletion named D17 that only removes sequences centromere-distal to the H1 domain also abolishes imprinting [28]. Experiments suggest that the essential factor Sap1 binds within this region, to the site named SAS1, and deletion of this site affects Sap1 binding in vitro, as well as imprinting in vivo. In addition, a linker-scanning mutagenesis study of the H1 domain identified a 6-bp substitution within the H1 domain called mut3 that reduces pausing at MPS1 [13]. Two others substitutions located within the part of the H1 domain that is covered by the smt0 deletion, named mut5 and mut7, also reduce imprinting. Characterization of the mut7 substitution suggested that it might affect the maintenance of the imprint throughout the cell cycle. During an earlier study done to characterize replication intermediates at the mat1 locus, replication initiation point (RIP) mapping was used to identify priming sites in the region. This method utilizes the fact that RNA primers on Okazaki fragments protect the fragments from degradation by l-exonuclease, allowing their selective purification and use for primer extension [29]. Using this method, we detected a strong priming site within the mat1M cassette, approximately 350 bp centromere-proximal to the imprint, but not at the site of the imprint itself [12]. A weak leading-strand replication pause site was also observed in this region. Here we identify and analyze the cis-acting sequences required for replication pausing and imprinting. Our results obtained by characterization of replication intermediates allow us to propose a mechanism for imprinting in fission yeast. Results Identification of two regions encoded by the M cassette that are required for imprinting and pausing In order to analyze the functional importance of the sequences within the mating-type cassette DNA, we utilized a strain that carried M information at the mat1 locus and was deleted for the two donor loci mat2P and mat3M. In this genetic background, MPS1 pausing and imprinting still occur, but the replication forks stalled at the imprint are resolved by sister chromatid recombination instead of recombination with the donor loci [30]. Thus, the M-cassette DNA is maintained at mat1, allowing mutations to be stably introduced at the locus. In an initial attempt to investigate the importance of the M-cassette sequence in pausing and imprinting, we deleted a 406-bp region starting just proximal to, but not including, where the two imprinted nucleotides are located and extending into the cassette DNA such that it ended within the Mc transcript encoded by the cassette (Figure 2A, strain SS4). Interestingly, analysis of this strain showed that the deletion abolished both imprinting, as detected by standard Southern blot analysis, and replication pausing at MPS1, as measured by 2D-gel electrophoresis (Figure 2B, 2C). Since this deletion covers a large region, it could potentially include several cis-acting sequences required for replication pausing and imprinting. Therefore, several shorter deletions within this region of interest were constructed (Figure 2). The 60-bp deletion in strain SS15, which included the previously mapped lagging-strand priming site (see Intro- duction), did not have an effect on pausing and only slightly reduced the level of imprinting. This deletion gave rise to 75.7% and 102% of wild-type levels of imprinting and PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1001328