Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast.

Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

The Smc5/6 complex is a DNA loop-extruding motor.

Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes1. Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown2. Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes.

Two pathways drive meiotic chromosome axis assembly in Saccharomyces cerevisiae.

Successful meiotic recombination, and thus fertility, depends on conserved axis proteins that organize chromosomes into arrays of anchored chromatin loops and provide a protected environment for DNA exchange. Here, we show that the stereotypic chromosomal distribution of axis proteins in Saccharomyces cerevisiae is the additive result of two independent pathways: a cohesin-dependent pathway, which was previously identified and mediates focal enrichment of axis proteins at gene ends, and a parallel cohesin-independent pathway that recruits axis proteins to broad genomic islands with high gene density. These islands exhibit elevated markers of crossover recombination as well as increased nucleosome density, which we show is a direct consequence of the underlying DNA sequence. A predicted PHD domain in the center of the axis factor Hop1 specifically mediates cohesin-independent axis recruitment. Intriguingly, other chromosome organizers, including cohesin, condensin, and topoisomerases, are differentially depleted from the same regions even in non-meiotic cells, indicating that these DNA sequence-defined chromatin islands exert a general influence on the patterning of chromosome structure.

Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus.

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis.

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.

Redirecting meiotic DNA break hotspot determinant proteins alters localized spatial control of DNA break formation and repair.

During meiosis, DNA double-strand breaks (DSBs) are formed at high frequency at special chromosomal sites, called DSB hotspots, to generate crossovers that aid proper chromosome segregation. Multiple chromosomal features affect hotspot formation. In the fission yeast S. pombe the linear element proteins Rec25, Rec27 and Mug20 are hotspot determinants - they bind hotspots with high specificity and are necessary for nearly all DSBs at hotspots. To assess whether they are also sufficient for hotspot determination, we localized each linear element protein to a novel chromosomal site (ade6 with lacO substitutions) by fusion to the Escherichia coli LacI repressor. The Mug20-LacI plus lacO combination, but not the two separate lac elements, produced a strong ade6 DSB hotspot, comparable to strong endogenous DSB hotspots. This hotspot had unexpectedly low ade6 recombinant frequency and negligible DSB hotspot competition, although like endogenous hotspots it manifested DSB interference. We infer that linear element proteins must be properly placed by endogenous functions to impose hotspot competition and proper partner choice for DSB repair. Our results support and expand our previously proposed DSB hotspot-clustering model for local control of meiotic recombination.

The Formation of Neochromosomes during Experimental Evolution in the Yeast Saccharomyces cerevisiae.

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

DNA Repair in Haploid Context.

DNA repair is a well-covered topic as alteration of genetic integrity underlies many pathological conditions and important transgenerational consequences. Surprisingly, the ploidy status is rarely considered although the presence of homologous chromosomes dramatically impacts the repair capacities of cells. This is especially important for the haploid gametes as they must transfer genetic information to the offspring. An understanding of the different mechanisms monitoring genetic integrity in this context is, therefore, essential as differences in repair pathways exist that differentiate the gamete's role in transgenerational inheritance. Hence, the oocyte must have the most reliable repair capacity while sperm, produced in large numbers and from many differentiation steps, are expected to carry de novo variations. This review describes the main DNA repair pathways with a special emphasis on ploidy. Differences between Saccharomyces cerevisiae and Schizosaccharomyces pombe are especially useful to this aim as they can maintain a diploid and haploid life cycle respectively.

Mechanism of MRX inhibition by Rif2 at telomeres.

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.

Sequence-dependent nucleosome formation in trinucleotide repeats evaluated by in vivo chemical mapping.

Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4-S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT)12 and (ACT)12 act as strong nucleosome-promoting sequences, while (AGG)12 and (CCG)12 act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG)12, is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.

Factors that influence bidirectional long-tract homozygosis due to double-strand break repair in Candida albicans.

Genomic rearrangements have been associated with the acquisition of adaptive phenotypes, allowing organisms to efficiently generate new favorable genetic combinations. The diploid genome of Candida albicans is highly plastic, displaying numerous genomic rearrangements that are often the by-product of the repair of DNA breaks. For example, DNA double-strand breaks (DSB) repair using homologous-recombination pathways are a major source of loss-of-heterozygosity (LOH), observed ubiquitously in both clinical and laboratory strains of C. albicans. Mechanisms such as break-induced replication (BIR) or mitotic crossover (MCO) can result in long tracts of LOH, spanning hundreds of kilobases until the telomere. Analysis of I-SceI-induced BIR/MCO tracts in C. albicans revealed that the homozygosis tracts can ascend several kilobases toward the centromere, displaying homozygosis from the break site toward the centromere. We sought to investigate the molecular mechanisms that could contribute to this phenotype by characterizing a series of C. albicans DNA repair mutants, including pol32-/-, msh2-/-, mph1-/-, and mus81-/-. The impact of deleting these genes on genome stability revealed functional differences between Saccharomyces cerevisiae (a model DNA repair organism) and C. albicans. In addition, we demonstrated that ascending LOH tracts toward the centromere are associated with intrinsic features of BIR and potentially involve the mismatch repair pathway which acts upon natural heterozygous positions. Overall, this mechanistic approach to study LOH deepens our limited characterization of DNA repair pathways in C. albicans and brings forth the notion that centromere proximal alleles from DNA break sites are not guarded from undergoing LOH.

Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double-strand break.

Chromatin remodeling is essential for effective repair of a DNA double-strand break (DSB). KAT5 (Schizosaccharomyces pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA DSB, including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1+ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1+ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination (HR). These phenotypes of mst1 are similar to pht1-4KR, a nonacetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs toward HR pathways by modulating resection at the DSB.

Functional control of Eco1 through the MCM complex in sister chromatid cohesion.

Sister chromatid cohesion (SCC) is essential for the maintenance of genome integrity. The establishment of SCC is coupled to DNA replication, and this is achieved in budding yeast Saccharomyces cerevisiae by a mechanism that is dependent on the interaction between Eco1 acetyltransferase and PCNA in the DNA replication complex. In vertebrates, the Eco1 homolog ESCO2 has been reported to interact with MCM complex in the DNA replication complex to establish DNA replication-dependent cohesion. Here we show that budding yeast Eco1 is also physically interacted with the MCM complex. We found that Eco1 was specifically bound to Mcm2 subunit in the MCM complex and they interacted via their N-terminal regions, using yeast two-hybrid system. The underlying mechanism of the interaction was different between yeast and vertebrates. Intensive molecular dissection of Eco1 identified residues important for interaction with Mcm2 and/or PCNA. Mutant forms of Eco1 (Eco1mWW and Eco1mGRK), where sets of the identified residues were substituted with alanine, resulted in impaired SCC, decreased level of acetylation of Smc3, and a reduction of Eco1 protein amount in yeast cells. We, hence, suggest that Eco1 is stabilized by its interactions with MCM complex and PCNA, which allows it to promote DNA replication-coupled SCC establishment.

Sir3 mediates long-range chromosome interactions in budding yeast.

Physical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here, we investigate the well-conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing SIR complex composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long-range contacts between distant regions, including the rDNA, telomeres, and internal Sir3-bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach, we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, Sir4), Sir2 activity, or chromosome context. Altogether, these data suggest that Sir3 acts as a molecular bridge that stabilizes long-range interactions.

Genetic and Molecular Approaches to Study Chromosomal Breakage at Secondary Structure-Forming Repeats.

DNA repeats capable of adopting stable secondary structures are hotspots for double-strand break (DSB) formation and, hence, for homologous recombination and gross chromosomal rearrangements (GCR) in many prokaryotic and eukaryotic organisms, including humans. Here, we provide protocols for studying chromosomal instability triggered by hairpin- and cruciform-forming palindromic sequences in the budding yeast, Saccharomyces cerevisiae. First, we describe two sensitive genetic assays aimed to determine the recombinogenic potential of inverted repeats and their ability to induce GCRs. Then, we detail an approach to monitor chromosomal DSBs by Southern blot hybridization. Finally, we describe how to define the molecular structure of DSBs. We provide, as an example, the analysis of chromosomal fragility at a reporter system containing unstable Alu-inverted repeats. By using these approaches, any DNA sequence motif can be assessed for its breakage potential and ability to drive genome instability.

14-3-3 Protein Bmh1 triggers short-range compaction of mitotic chromosomes by recruiting sirtuin deacetylase Hst2.

During mitosis, chromosomes are compacted in length by more than 100-fold into rod-shaped forms. In yeast, this process depends on the presence of a centromere, which promotes condensation in cis by recruiting mitotic kinases such as Aurora B kinase. This licensing mechanism enables the cell to discriminate chromosomal from noncentromeric DNA and to prohibit the propagation of the latter. Aurora B kinase elicits a cascade of events starting with phosphorylation of histone H3 serine 10 (H3S10ph), which signals the recruitment of lysine deacetylase Hst2 and the removal of lysine 16 acetylation in histone 4. The unmasked histone 4 tails interact with the acidic patch of neighboring nucleosomes to drive short-range compaction of chromatin, but the mechanistic details surrounding the Hst2 activity remain unclear. Using in vitro and in vivo assays, we demonstrate that the interaction of Hst2 with H3S10ph is mediated by the yeast 14-3-3 protein Bmh1. As a homodimer, Bmh1 binds simultaneously to H3S10ph and the phosphorylated C-terminus of Hst2. Our pull-down experiments with extracts of synchronized cells show that the Hst2-Bmh1 interaction is cell cycle dependent, peaking in the M phase. Furthermore, we show that phosphorylation of C-terminal residues of Hst2, introduced by genetic code expansion, stimulates its deacetylase activity. Hence, the data presented here identify Bmh1 as a key player in the mechanism of licensing of chromosome compaction in mitosis.

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.

The role of gene dosage in budding yeast centrosome scaling and spontaneous diploidization.

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids 'rescues' SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes.

Fission yeast condensin contributes to interphase chromatin organization and prevents transcription-coupled DNA damage.

BACKGROUND: Structural maintenance of chromosomes (SMC) complexes are central organizers of chromatin architecture throughout the cell cycle. The SMC family member condensin is best known for establishing long-range chromatin interactions in mitosis. These compact chromatin and create mechanically stable chromosomes. How condensin contributes to chromatin organization in interphase is less well understood. RESULTS: Here, we use efficient conditional depletion of fission yeast condensin to determine its contribution to interphase chromatin organization. We deplete condensin in G2-arrested cells to preempt confounding effects from cell cycle progression without condensin. Genome-wide chromatin interaction mapping, using Hi-C, reveals condensin-mediated chromatin interactions in interphase that are qualitatively similar to those observed in mitosis, but quantitatively far less prevalent. Despite their low abundance, chromatin mobility tracking shows that condensin markedly confines interphase chromatin movements. Without condensin, chromatin behaves as an unconstrained Rouse polymer with excluded volume, while condensin constrains its mobility. Unexpectedly, we find that condensin is required during interphase to prevent ongoing transcription from eliciting a DNA damage response. CONCLUSIONS: In addition to establishing mitotic chromosome architecture, condensin-mediated long-range chromatin interactions contribute to shaping chromatin organization in interphase. The resulting structure confines chromatin mobility and protects the genome from transcription-induced DNA damage. This adds to the important roles of condensin in maintaining chromosome stability.

Loss of FZO1 gene results in changes of cell dynamics in fission yeast.

Mitochondrial fission and fusion dynamics are critical cellular processes, and abnormalities in these processes are associated with severe human disorders, such as Beckwith­Wiedemann syndrome, neurodegenerative diseases, Charcot­Marie­Tooth disease type 6, multiple symmetric lipomatosis and microcephaly. Fuzzy onions protein 1 (Fzo1p) regulates mitochondrial outer membrane fusion. In the present study, Schizosaccharomyces pombe (S. pombe) was used to explore the effect of FZO1 gene deletion on cell dynamics in mitosis. The mitochondrial morphology results showed that the mitochondria appeared to be fragmented and tubular in wild­type cells; however, they were observed to accumulate in fzo1Δ cells. The FZO1 gene deletion was demonstrated to result in slow proliferation, sporogenesis defects, increased microtubule (MT) number and actin contraction defects in S. pombe. The FZO1 gene deletion also affected the rate of spindle elongation and phase time at the metaphase and anaphase, as well as spindle MT organization. Live­cell imaging was performed on mutant strains to observe three distinct kinetochore behaviors (normal, lagging and mis­segregation), as well as abnormal spindle breakage. The FZO1 gene deletion resulted in coenzyme and intermediate metabolite abnormalities as determined via metabolomics analysis. It was concluded that the loss of FZO1 gene resulted in deficiencies in mitochondrial dynamics, which may result in deficiencies in spindle maintenance, chromosome segregation, spindle breakage, actin contraction, and coenzyme and intermediate metabolite levels.