Identification of a Novel Type of Spacer Element Required for Imprinting in Fission Yeast Suha Sayrac, Sonya Vengrova, Emma L. Godfrey, Jacob Z. Dalgaard* Clinical Sciences Research Institute, Gibbet Hill Campus, University of Warwick, Coventry, United Kingdom Abstract Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging- strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully- processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers. Citation: Sayrac S, Vengrova S, Godfrey EL, Dalgaard JZ (2011) Identification of a Novel Type of Spacer Element Required for Imprinting in Fission Yeast. PLoS Genet 7(3): e1001328. doi:10.1371/journal.pgen.1001328 Editor: James E. Haber, Brandeis University, United States of America Received July 15, 2010; Accepted February 7, 2011; Published March 10, 2011 Copyright: 2011 Sayrac et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from Marie Curie Cancer Care. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.z.dalgaard@warwick.ac.uk Introduction Differentiated sister chromatids, coupled with non-random segregation, have been proposed to control cell fate during the development and differentiation of multicellular organisms [1]. Similarly, cellular differentiation in the fission yeast Schizosacchar- omyces pombe involves the establishment of differentiated chromatids and their asymmetrical segregation. The nature of this epigenetic mark or imprint that acts to discriminate the chromatids has been defined, however, the mechanism that leads to its introduction remains unknown. Fission yeast cells exhibit two different mating- types denoted as M and P. Homothallic strains are able to switch between mating-types in a pattern where only one granddaughter of a newly switched cell will have switched mating-type (Figure 1A) [2–6]. The mating-type of a cell is determined by the transcriptionally expressed mat1 locus located on chromosome II, which encodes either an M- or a P-specific gene cassette (Figure 1B) [7,8]. Switching between the two mating-types occurs through a recombination event that replaces the cassette present at mat1 with that of the opposite mating-type, using one of the two transcriptionally silenced loci, mat2P and mat3M, as a donor of the information. The molecular mechanism underlying mating-type switching is tightly linked to DNA replication, and the asymmetrical pattern of switching reflects the asymmetry of the replication process. The mat1 locus is replicated unidirectionally in the centromere- proximal direction (see below). An imprint, which marks the mat1 locus of switchable cells (Figure 1B, 1C), is introduced during lagging-strand synthesis of the mat1 DNA in only one of the two sister chromatids formed in S phase [9,10]. This imprint is maintained for one cell cycle, then during the next S phase, the imprint acts as a barrier for leading-strand replication, resulting in the replication-coupled recombination event that underlies mating-type switching [11–13]. The recombination event pro- gresses by the ‘‘synthesis dependent single-strand annealing mechanism’’ where only one DNA strand at the donor locus provides the genetic information that is transferred to mat1 [14]. The homology needed for both the initiation and resolution of this recombination event is provided by short homology domains, named H1 and H2, that flank both the mat1 locus and each of the two donor loci, mat2P and mat3M (Figure 1B) [8,15]. As a result of this recombination event, the mating-type cassette of only one of the two sister chromatids is replaced by a cassette containing the opposite mating-type information, such that the subsequent cell division gives rise to two cells with opposite mating-types. The imprint was first identified genetically as a strand-specific, chromosomally inherited mark that is linked to the mat1 locus and is required for the induction of switching [4–6,16]. Subsequently it was shown that the imprint consists of two ribonucleotides that are incorporated into the DNA duplex in a strand- and site-specific manner ([12] and reviewed in [17]). These two ribonucleotides are located at the junction between the mating-type cassette and the homology domain H1 (Figure 1B) [18]. The bonds of the RNA- DNA hybrid at the imprint site appear to be efficiently hydrolyzed by cellular enzymes, making purification of intact imprinted DNA difficult. During standard purification methods, the imprint is converted into a double-stranded break that can be detected by Southern analysis [10,19]. However, we have managed to purify PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1001328