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, which suggests 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 budding yeast of 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.

OriDB (http://www.oridb.org/) is a database containing collated genome-wide mapping studies of confirmed and predicted replication origin sites. The original database collated and curated Saccharomyces cerevisiae origin mapping studies. Here, we report that the OriDB database and web site have been revamped to improve user accessibility to curated data sets, to greatly increase the number of curated origin mapping studies, and to include the collation of replication origin sites in the fission yeast Schizosaccharomyces pombe. The revised database structure underlies these improvements and will facilitate further expansion in the future. The updated OriDB for S. cerevisiae is available at http://cerevisiae.oridb.org/ and for S. pombe at http://pombe.oridb.org/.

Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.

Genome-wide DNA polymerase usage maps determined in fission yeast, using a new sequencing strategy based on ribonucleotide misincorporation, track the division of labor between replicative polymerases and reveal locations and efficiencies of replication origins. Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α–primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ.

When all origins of replication are deleted from the archaeon Haloferax volcanii, homologous recombination is used to initiate DNA replication and the growth rate is accelerated. Duplication of DNA genomes in bacteria, archaea and eukaryotes initiates at sites called origins of replication. Some viruses, however, lack such origins and instead replicate by a recombination-dependent mechanism. Thorsten Allers and colleagues now report the first example of an archaeon that is able to replicate when all of its origins are deleted. As expected, deletion of a subset of Haloferax volcanii's four origins resulted in slower growth. But surprisingly, when all the origins were deleted, cell growth was actually faster than in the wild type. These cells are able to use recombination, initiating at disperse sites, to replicate a genome that is much larger than that found in viruses. This prompts the question, for what purpose did replication origins evolve? DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life1 and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whereas most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We use deep sequencing to study replication in Haloferax volcanii and identify four chromosomal origins of differing activity. Deletion of individual origins results in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.

INTRODUCTION: Although much effort has been devoted to studying yeast in the past few decades, our understanding of this model organism is still limited. Rapidly developing DNA synthesis techniques have made a “build-to-understand” approach feasible to reengineer on the genome scale. Here, we report on the completion of a 770-kilobase synthetic yeast chromosome II (synII). SynII was characterized using extensive Trans-Omics tests. Despite considerable sequence alterations, synII is virtually indistinguishable from wild type. However, an up-regulation of translational machinery was observed and can be reversed by restoring the transfer RNA (tRNA) gene copy number.RATIONALE: Following the “design-build-test-debug” working loop, synII was successfully designed and constructed in vivo. Extensive Trans-Omics tests were conducted, including phenomics, transcriptomics, proteomics, metabolomics, chromosome segregation, and replication analyses. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification byloxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium.RESULTS: To efficiently construct megabase-long chromosomes, we developed an I-SceI–mediated strategy, which enables parallel integration of synthetic chromosome arms and reduced the overall integration time by 50% for synII. An I-SceI site is introduced for generating a double-strand break to promote targeted homologous recombination during mitotic growth. Despite hundreds of modifications introduced, there are still regions sharing substantial sequence similarity that might lead to undesirable meiotic recombinations when intercrossing the two semisynthetic chromosome arm strains. Induction of the I-SceI–mediated double-strand break is otherwise lethal and thus introduced a strong selective pressure for targeted homologous recombination. Since our strategy is designed to generate a markerless synII and leave theURA3marker on the wild-type chromosome, we observed a tenfold increase inURA3-deficient colonies upon I-SceI induction, meaning that our strategy can greatly bias the crossover events toward the designated regions.By incorporating comprehensive phenotyping approaches at multiple levels, we demonstrated that synII was capable of powering the growth of yeast indistinguishably from wild-type cells (see the figure), showing highly consistent biological processes comparable to the native strain. Meanwhile, we also noticed modest but potentially significant up-regulation of the translational machinery. The main alteration underlying this change in expression is the deletion of 13 tRNA genes.A growth defect was observed in one very specific condition—high temperature (37°C) in medium with glycerol as a carbon source—where colony size was reduced significantly. We targeted and debugged this defect by two distinct approaches. The first approach involved phenotype screening of all intermediate strains followed by a complementation assay with wild-type sequences in the synthetic strain. By doing so, we identified a modification resulting from PCRTag recoding inTSC10, which is involved in regulation of the yeast high-osmolarity glycerol (HOG) response pathway. After replacement with wild-typeTSC10, the defect was greatly mitigated. The other approach, debugging by SCRaMbLE, showed rearrangements in regions containing HOG regulation genes. Both approaches indicated that the defect is related to HOG response dysregulation. Thus, the phenotypic defect can be pinpointed and debugged through multiple alternative routes in the complex cellular interactome network.CONCLUSION: We have demonstrated that synII segregates, replicates, and functions in a highly similar fashion compared with its wild-type counterpart. Furthermore, we believe that the iterative “design-build-test-debug” cycle methodology, established here, will facilitate progression of the Sc2.0 project in the face of the increasing synthetic genome complexity. SynII characterization.: (A) Cell cycle comparison between synII and BY4741 revealed by the percentage of cells with separated CEN2-GFP dots, metaphase spindles, and anaphase spindles. (B) Replication profiling of synII (red) and BY4741 (black) expressed as relative copy number by deep sequencing. (C) RNA sequencing analysis revealed that the significant up-regulation of translational machinery in synII is induced by the deletion of tRNA genes in synII.