Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation-sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex-like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex-like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.

Molecular biology has been led by various measurement technologies, and increased throughput has developed omics analysis. The development of massively parallel sequencing technology has enabled access to fundamental molecular data and revealed genomic and transcriptomic signatures. Nanopore sequencers have driven such evolution to the next stage. Oxford Nanopore Technologies Inc. provides a new type of single molecule sequencer using protein nanopore that realizes direct sequencing without DNA synthesizing or amplification. This nanopore sequencer can sequence an ultra‐long read limited by the input nucleotide length, or can determine DNA/RNA modifications. Recently, many fields such as medicine, epidemiology, ecology, and education have benefited from this technology. In this review, we explain the features and functions of the nanopore sequencer, introduce various situations where it has been used as a critical technology, and expected future applications.

Members of the family Araneidae are common orb-weaving spiders, and they produce several types of silks throughout their behaviors and lives, from reproduction to foraging. Egg sac, prey capture thread, or dragline silk possesses characteristic mechanical properties, and its variability makes it a highly attractive material for ecological, evolutional, and industrial fields. However, the complete set of constituents of silks produced by a single species is still unclear, and novel spidroin genes as well as other proteins are still being found. Here, we present the first genome in genus Araneus together with the full set of spidroin genes with unamplified long reads and confirmed with transcriptome of the silk glands and proteome analysis of the dragline silk. The catalogue includes the first full length sequence of a paralog of major ampullate spidroin MaSp3 , and several spider silk-constituting elements designated SpiCE. Family-wide phylogenomic analysis of Araneidae suggests the relatively recent acquisition of these genes, and multiple-omics analyses demonstrate that these proteins are critical components in the abdominal spidroin gland and dragline silk, contributing to the outstanding mechanical properties of silk in this group of species.

Spider silk fiber rapidly assembles from spidroin protein in soluble state via an incompletely understood mechanism. Here, we present an integrated model for silk formation that incorporates the effects of multiple chemical and physical gradients on the different spidroin functional domains. Central to the process is liquid-liquid phase separation (LLPS) that occurs in response to multivalent anions such as phosphate, mediated by the carboxyl-terminal and repetitive domains. Acidification coupled with LLPS triggers the swift self-assembly of nanofibril networks, facilitated by dimerization of the amino-terminal domain, and leads to a liquid-to-solid phase transition. Mechanical stress applied to the fibril structures yields macroscopic fibers with hierarchical organization and enriched for β-sheet conformations. Studies using native silk gland material corroborate our findings on spidroin phase separation. Our results suggest an intriguing parallel between silk assembly and other LLPS-mediated mechanisms, such as found in intracellular membraneless organelles and protein aggregation disorders.

Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining high-quality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders. We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.

BackgroundDuring the replication process of bacteria with circular chromosomes, an odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. However, many bacteria harbor a conserved dimer resolution machinery consisting of one or two tyrosine recombinases, XerC and XerD, and their 28-bp target site, dif.ResultsTo study the evolution of the dif/XerCD system and its relationship with replication termination, we report the comprehensive prediction of dif sequences in silico using a phylogenetic prediction approach based on iterated hidden Markov modeling. Using this method, dif sites were identified in 641 organisms among 16 phyla, with a 97.64% identification rate for single-chromosome strains. The dif sequence positions were shown to be strongly correlated with the GC skew shift-point that is induced by replicational mutation/selection pressures, but the difference in the positions of the predicted dif sites and the GC skew shift-points did not correlate with the degree of replicational mutation/selection pressures.ConclusionsThe sequence of dif sites is widely conserved among many bacterial phyla, and they can be computationally identified using our method. The lack of correlation between dif position and the degree of GC skew suggests that replication termination does not occur strictly at dif sites.

BackgroundBiochemical pathways provide an essential context for understanding comprehensive experimental data and the systematic workings of a cell. Therefore, the availability of online pathway browsers will facilitate post-genomic research, just as genome browsers have contributed to genomics. Many pathway maps have been provided online as part of public pathway databases. Most of these maps, however, function as the gateway interface to a specific database, and the comprehensiveness of their represented entities, data mapping capabilities, and user interfaces are not always sufficient for generic usage.Methodology/Principal FindingsWe have identified five central requirements for a pathway browser: (1) availability of large integrated maps showing genes, enzymes, and metabolites; (2) comprehensive search features and data access; (3) data mapping for transcriptomic, proteomic, and metabolomic experiments, as well as the ability to edit and annotate pathway maps; (4) easy exchange of pathway data; and (5) intuitive user experience without the requirement for installation and regular maintenance. According to these requirements, we have evaluated existing pathway databases and tools and implemented a web-based pathway browser named Pathway Projector as a solution.Conclusions/SignificancePathway Projector provides integrated pathway maps that are based upon the KEGG Atlas, with the addition of nodes for genes and enzymes, and is implemented as a scalable, zoomable map utilizing the Google Maps API. Users can search pathway-related data using keywords, molecular weights, nucleotide sequences, and amino acid sequences, or as possible routes between compounds. In addition, experimental data from transcriptomic, proteomic, and metabolomic analyses can be readily mapped. Pathway Projector is freely available for academic users at http://www.g-language.org/PathwayProjector/.

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.