Benoit Arcangioli

RNAi promotes heterochromatic silencing through replication-coupled release of RNA Pol II

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification.

A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast

Mating‐type switching in the fission yeast Schizosaccharomyces pombe results in the transfer of genetic information from one of the two silent cassettes (mat2P or mat3M) to the transcriptionally active locus (mat1). The switching pattern is programmed by an imprinting event which restricts mat1 gene conversion to only one of the two sister cells, leading to asymmetric cell division. Biochemical analysis indicated that the mat1 locus contains a fragile chromosomal site. Southern hybridization and primer extension experiments showed that the fragility consists of a single‐strand break (SSB). The nicked DNA is stable throughout the cell cycle. The features of the nick fulfil all the requirements for the ‘epigenetic’, site and strand‐specific chromosome modification at the mat1 locus, providing strong evidence that an SSB can initiate mitotic and meiotic gene conversion during replication.

Mus81 is essential for sister chromatid recombination at broken replication forks

Recombination is essential for the recovery of stalled/collapsed replication forks and therefore for the maintenance of genomic stability. The situation becomes critical when the replication fork collides with an unrepaired single‐strand break and converts it into a one‐ended double‐strand break. We show in fission yeast that a unique broken replication fork requires the homologous recombination (HR) enzymes for cell viability. Two structure‐specific heterodimeric endonucleases participate in two different resolution pathways. Mus81/Eme1 is essential when the sister chromatid is used for repair; conversely, Swi9/Swi10 is essential when an ectopic sequence is used for repair. Consequently, the utilization of these two HR modes of resolution mainly relies on the ratio of unique and repeated sequences present in various eukaryotic genomes. We also provide molecular evidence for sister recombination intermediates. These findings demonstrate that Mus81/Eme1 is the dedicated endonuclease that resolves sister chromatid recombination intermediates during the repair of broken replication forks.

Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance

Sumoylation represents a conserved mechanism of post‐translational protein modification. We report that Pli1p, the unique fission yeast member of the SP‐RING family, is a SUMO E3 ligase in vivo and in vitro. pli1Δ cells display no obvious mitotic growth defects, but are sensitive to the microtubule‐destabilizing drug TBZ and exhibit enhanced minichromosome loss. The weakened centromeric function of pli1Δ cells may be related to the defective heterochromatin structure at the central core, as shown by the reduced silencing of an ura4 variegation reporter gene inserted at cnt and imr. Interestingly, pli1Δ cells also exhibit enhanced loss of the ura4 reporter at these loci, likely by gene conversion using homologous sequences as information donors. Moreover, pli1Δ cells exhibit consistent telomere length increase, possibly achieved by a similar process. Point mutations within the RING finger of Pli1p totally or partially reproduce the pli1 deletion phenotypes, thus correlating with their sumoylation activity. Altogether, these results strongly suggest that Pli1p, and by extension sumoylation, is involved in mechanisms that regulate recombination in particular heterochromatic repeated sequences.

Fission yeast switches mating type by a replication–recombination coupled process

Fission yeast exhibits a homothallic life cycle, in which the mating type of the cell mitotically alternates in a highly regulated fashion. Pedigree analysis of dividing cells has shown that only one of the two sister cells switches mating type. It was shown recently that a site‐ and strand‐specific DNA modification at the mat1 locus precedes mating‐type switching. By tracking the fate of mat1 DNA throughout the cell cycle with a PCR assay, we identified a novel DNA intermediate of mating‐type switching in S‐phase. The time and rate of appearance and disappearance of this DNA intermediate are consistent with a model in which mating‐type switching occurs through a replication–recombination coupled pathway. Such a process provides experimental evidence in support of a copy choice recombination model in Schizosaccharomyces pombe mating‐type switching and is reminiscent of the sister chromatid recombination used to complete replication in the presence of certain types of DNA damage.

CENP-B preserves genome integrity at replication forks paused by retrotransposon LTR

Centromere-binding protein B (CENP-B) is a widely conserved DNA binding factor associated with heterochromatin and centromeric satellite repeats. In fission yeast, CENP-B homologues have been shown to silence long terminal repeat (LTR) retrotransposons by recruiting histone deacetylases. However, CENP-B factors also have unexplained roles in DNA replication. Here we show that a molecular function of CENP-B is to promote replication-fork progression through the LTR. Mutants have increased genomic instability caused by replication-fork blockage that depends on the DNA binding factor switch-activating protein 1 (Sap1), which is directly recruited by the LTR. The loss of Sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. We conclude that retrotransposons influence replication polarity through recruitment of Sap1 and transposition near replication-fork blocks, whereas CENP-B counteracts this activity and promotes fork stability. Our results may account for the role of LTR in fragile sites, and for the association of CENP-B with pericentromeric heterochromatin and tandem satellite repeats.

Regulation of Histone H3 Lysine 56 Acetylation in Schizosaccharomyces pombe

In Saccharomyces cerevisiae, acetylation of lysine 56 (Lys-56) in the globular domain of histone H3 plays an important role in response to genotoxic agents that interfere with DNA replication. However, the regulation and biological function of this modification are poorly defined in other eukaryotes. Here we show that Lys-56 acetylation in Schizosaccharomyces pombe occurs transiently during passage through S-phase and is normally removed in G2. Genotoxic agents that cause DNA double strand breaks during replication elicit a delay in deacetylation of histone H3 Lys-56. In addition, mutant cells that cannot acetylate Lys-56 are acutely sensitive to genotoxic agents that block DNA replication. Moreover, we show that Spbc342.06cp, a previously uncharacterized open reading frame, encodes the functional homolog of S. cerevisiae Rtt109, and that this protein acetylates H3 Lys-56 both in vitro and in vivo. Altogether, our results indicate that both the regulation of histone H3 Lys-56 acetylation by its histone acetyltransferase and histone deacetylase and its role in the DNA damage response are conserved among two distantly related yeast model organisms.

Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51‐dependent recombination outcomes

Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5‐GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Δ mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Δ mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage‐induced Rhp51 foci. The number of UV‐induced Rhp51 foci is partially reduced in swi5Δ and rhp57Δ mutants and completely abolished in an swi5Δ rhp57Δ double mutant. An assay for products generated by HO endonuclease‐induced DNA double‐strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.

Role of SUMO in the dynamics of telomere maintenance in fission yeast

The sheltering of chromosome ends from illegitimate DNA repair reactions and telomere length homeostasis are critical for preserving genomic integrity. Growing evidence implicates covalent protein modification by SUMO (small ubiquitin-like modifier) (sumoylation) in the regulation of numerous DNA transactions, including DNA repair and transcription, as well as heterochromatin formation and maintenance. We have recently shown that fission yeast Pli1p is a SUMO E3 ligase and that pli1 mutants, which are impaired for global sumoylation, are viable, but exhibit de-regulated homologous recombination and marked defects in chromosome segregation and centromeric silencing, as well as a consistent increase in telomere length. In this work, we explore the mechanisms underlying sumoylation-dependent telomere maintenance. We show that Pli1p, but not the related Nse2p, is the principal SUMO E3 ligase enzyme involved. Using both a pli1 mutation and a physiological “knockdown” of sumoylation, achieved by inducible expression of a dominant negative form of the conjugating enzyme Ubc9p, we further show that telomere lengthening induced by lack of sumoylation is not due to unscheduled telomere–telomere recombination. Instead, sumoylation increases telomerase activity, therefore suggesting that this modification controls the activity of a positive or negative regulator of telomerase.

Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast

Mating‐type switching in the fission yeast Schizosaccharomyces pombe is initiated by a strand‐specific imprint located at the mating‐type (mat1) locus. We show that the imprint corresponds to a single‐strand DNA break (SSB), which is site‐ but not sequence‐specific. We identified three novel cis‐acting elements, involved in the formation and stability of the SSB. One of these elements is essential for a replication fork pause next to mat1 and interacts in vivo with the Swi1 protein. Another element is essential for maintaining the SSB during cell cycle progression. These results suggest that the DNA break appears during the S‐phase and is actively protected against repair. Consequently, during the following round of replication, a polar double‐strand break is formed. We show that when the replication fork encounters the SSB, the leading‐strand DNA polymerase is able to synthesize DNA to the edge of the SSB, creating a blunt‐ended recombination intermediate.