Replication Pausing and Imprinting Figure 1. Mating-type switching in S. pombe. (A) Switching pedigree. The cells of minus (M) and plus (P) mating type are represented with light and dark brown colours, respectively. Division of an unswitchable (u) cell leads to the formation of an unswitchable and a switchable (s) cell of the same mating type. Division of the switchable cell gives rise to a switchable cell of the same mating type and an unswitchable cell of the opposite mating type. (B) The mating-type region on chromosome II. The transcriptionally active mat1 locus and the silenced donor loci, mat2P and mat3M, are shown. The M and P information is indicated with light and dark brown colours, respectively. The homology domains H1, H2 and H3 flanking the loci are represented by grey boxes. The centromere (cen2) and the origin of replication located centromere-distal to mat1 are shown with a hollow box and a circle, respectively. The replication termination element RTS1 is indicated with grey triangles, and direction of fork movement through mat1 is shown above with grey arrows, representing unidirectional replication. The imprint is indicated by a yellow star. (C) The replication of the mat1 locus in unswitchable and switchable cells. The nascent leading and lagging strands are represented by red and blue lines, respectively. In the second generation, the inherited template strands are also shown in red or blue, depending on which strand they were replicated as during the S phase of the parent. The polarities of DNA strands are indicated with 39 and 59 symbols. The S phases of both cell types involves pausing of the replication fork at mat1 and the introduction of the imprint in the newly synthesized upper strand. In addition, during the S phase of a switchable cell, the imprint on the upper template strand acts as a lesion to block leading-strand synthesis. The stalled leading-strand replication complex induces homologous recombination between mat1 and the donor loci leading to mating-type switching. doi:10.1371/journal.pgen.1001328.g001 pausing, respectively. Extending this deletion a further 60 bp centromere-proximal into the M cassette (strain SS14) also had little effect on pausing and imprinting, implying that this region is not required for these processes. However, deletion of the remaining centromere-distal 316-bp region (strain SS3) abol- ished both imprinting and replication pausing, establishing that cis-acting sequences required for both these processes are located within this region. To further define the location of these cis-acting sequences, the 316-bp region was divided into two, deleting either the 205-bp centromere-proximal segment or the 111-bp centromere-distal segment separately (Figure 2, strains SS13 & SS2, respectively). Surprisingly, deletion of the centromere-proximal segment completely abolished imprinting but only slightly affected replication pausing. Deletion of the centromere-distal part however, completely abolished both imprinting and replication pausing. Therefore, this initial analysis identified two regions within the M cassette that are of importance for the imprinting process in different ways. PLoS Genetics | www.plosgenetics.org 3 March 2011 | Volume 7 | Issue 3 | e1001328