Dr. Sonya Vengrova
RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching.
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.
- RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching (Abstract on PubMed)
- The wild-type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides (Abstract on PubMed)
Dr. Suha Sayrac
Identification of a novel type of spacer element required for imprinting in fission yeast.
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 required for the asymmetrical switching pattern of S. pombe is known.
We have earlier 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 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 is unknown.
Here we present key novel discoveries important for unraveling the mechanism. Firstly, we define the sequences necessary for pausing the replication fork at the site of imprinting. Secondly, we show that there are no sequence requirements to the imprinted nucleotides as well as the region surrounding them. Thirdly, we define a novel spacer element that is required for imprinting and that affects priming sites after the replication fork is released from the replication pause.
Dr. Milana Koulintchenko
The S. pombe Pol-a mutation swi7 that abolishes imprinting at the mating-type locus also confers genetic instability and alkylation DNA damage sensitivity.
Polymerase a is an essential enzyme mostly involved in lagging strand replication where it mediates Okazaki fragment synthesis. Interestingly, a point mutation, named swi7, has been isolated in Schizosaccharomyces pombe polymerase a. swi7 abolishes the imprinting required for mating-type switching without affecting the essential function of the polymerase.
Here we investigate whether this mutation confers any genome wide defects. We show that the swi7 mutation renders cells hypersensitive to the DNA damaging agents methyl metansulfonate (MMS), hydroxyurea (HU) and UV. In addition we show that there is an increased number of Rad22 foci and hyper recombination in an ade6 recombination assay, indicative of increased genetic instability in the swi7 background. These data suggest that the swi7 mutation affects the general replication process.
Furthermore, we also detect novel swi1, swi3 and swi7 dependent alkylation damage repair intermediates, suggesting that swi7 might have a direct role in alkylation damage repair. An epitasis analysis shows that rad2 (Fen1) is epistatic with swi7, but not with swi1 and swi3, with regards to MMS sensitivity. The novel repair intermediates are also absent from MMS treated rad2 cells. We discuss the possibility that imprints similar to the mat1 imprint might be introduced at damaged bases during the replication process.
Dr. Tomoko Yamada-Inagawa
Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism.
Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event.
A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.
Dr. Takabumi Inagawa
Schizosaccharomyces pombe Rtf2 mediates site-specific replication termination by inhibiting replication restart.
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.
Dr. Elena Sommariva
Dr. Till Pellny
Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage.
The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1.
This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage.
Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.
- Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage (Abstract on PubMed)
Dr. Sandra Codlin
Complex mechanism of site-specific DNA replication termination in fission yeast.
A site-specific replication terminator, RTS1, is present at the Schizosaccharomyces pombe mating-type locus mat1. RTS1 regulates the direction of replication at mat1, optimizing mating-type switching that occurs as a replication-coupled recombination event. Here we show that RTS1 contains two cis-acting sequences that cooperate for efficient replication termination.
First, a sequence of approximately 450 bp containing four repeated 55 bp motifs is essential for function. Secondly, a purine-rich sequence of approximately 60 bp without intrinsic activity, located proximal to the repeats, acts cooperatively to increase barrier activity 4-fold. Our data suggest that the trans-acting factors rtf1p and rtf2p act through the repeated motifs and the purine-rich element, respectively. Thus, efficient site-specific replication termination at RTS1 occurs by a complex mechanism involving several cis-acting sequences and trans-acting factors. Interestingly, RTS1 displays similarities to mammalian rDNA replication barriers.
- Complex mechanism of site-specific DNA replication termination in fission yeast. (Abstract on PubMed)
M. Sc. Trevor Eydmann
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.