Identification of a novel type of spacer element required for imprinting in fission yeast

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 laggingstrand 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-fullyprocessed 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

Polymerase δ replicates both strands after homologous recombination-dependent fork restart

To maintain genetic stability DNA must be replicated only once and replication completed even when individual replication forks are inactivated. Because fork inactivation is common, the passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via Homologous Recombination Restarted Replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis caused by HoRReR being more error prone than canonical replication. This increased error rate implies that the HoRReR mechanism is distinct from that of a canonical fork. Here we exploit the fission yeast Schizosaccharomyces pombe to demonstrate that a DNA sequence duplicated by HoRReR during S phase is replicated semi-conservatively, but that both the leading and lagging strands are synthesised by DNA polymerase delta

Schizosaccharomyces pombe Rtf2 mediates site-specific replication termination by inhibiting replication restart

Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of “slow-moving” Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin

Rtf1-mediated eukaryotic site-specific replication termination

The molecular mechanisms mediating eukaryotic replication termination and pausing remain largely unknown. Here we present the molecular characterization of Rtf1 that mediates site-specific replication termination at the polar Schizosaccharomyces pombe barrier RTS1. We show that Rtf1 possesses two chimeric myb/SANT domains: one is able to interact with the repeated motifs encoded by the RTS1 element as well as the elements enhancer region, while the other shows only a weak DNA binding activity. In addition we show that the C-terminal tail of Rtf1 mediates self-interaction, and deletion of this tail has a dominant phenotype. Finally, we identify a point mutation in Rtf1 domain I that converts the RTS1 element into a replication barrier of the opposite polarity. Together our data establish that multiple protein DNA and protein–protein interactions between Rtf1 molecules and both the repeated motifs and the enhancer region of RTS1 are required for site-specific termination at the RTS1 element