The fission yeast clade, comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus and S. japonicus, occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, suggesting a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

Mating-type switching in fission yeast depends on an imprint at the mat1 locus. Previous data showed that the imprint is made in the DNA strand replicated as lagging. We now identify this imprint as an RNase-sensitive modification and suggest that it consists of one or two RNA residues incorporated into the mat1 DNA. Formation of the imprint requires swi1-and swi3-dependent pausing of the replication fork. Interestingly, swi1 and swi3 mutations that abolish pausing do not affect the use of lagging-strand priming site during replication. We show that the pausing of replication and subsequent formation of the imprint occur after the leading-strand replication complex has passed the site of the imprint and after lagging-strand synthesis has initiated at this proximal priming site. We propose a model in which a swi1-and swi3-dependent signal during lagging-strand synthesis leads to pausing of leading-strand replication and the introduction of the imprint.[Keywords: DNA modification; imprint; replication pausing; mating-type switching; Schizosaccharomyces pombe] Supplemental material is available at http://www.genesdev.org.

The imprint at the mat1 locus of Schizosaccharomyces pombe acts to initiate the replication-coupled recombination event that underlies mating-type switching. However, the nature of the imprint has been an area of dispute. Two alternative models have been proposed: one stated that the imprint is a nick in the DNA, whereas our data suggested that it consists of one or two ribonucleotides incorporated into the otherwise intact DNA duplex. Here, we verify key predictions of the RNA model by characterization of wild-type genomic DNA purified under conditions known to hydrolyse DNA-RNA-DNA hybrid strands. First, we observe one-nucleotide gap at the hydrolysed DNA, as expected from the presence of two ribonucleotides. Second, using a novel assay based on ligation-mediated PCR, a 3 0 -terminal ribonucleotide is detected at the hydrolysed imprint. Our observations allow the unification of available data sets characterizing the wild-type imprint.

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.

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