Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk-, with the complexes failing to associate with chromosomes.

Knowledge of protein-DNA binding specificity has important implications in understanding DNA metabolism, transcriptional regulation and developing therapeutic drugs. Previous studies demonstrated hydrogen bonds between amino acid side chains and DNA bases play major roles in specific protein-DNA interactions. In this paper, we investigated the roles of individual DNA strands and protein secondary structure types in specific protein-DNA recognition based on side chain-base hydrogen bonds. By comparing the contribution of each DNA strand to the overall binding specificity between DNA-binding proteins with different degrees of binding specificity, we found that highly specific DNA-binding proteins show balanced hydrogen bonding with each of the two DNA strands while multi-specific DNA binding proteins are generally biased towards one strand. Protein-base pair hydrogen bonds, in which both bases of a base pair are involved in forming hydrogen bonds with amino acid side chains, are more prevalent in the highly specific protein-DNA complexes than those in the multi-specific group. Amino acids involved in side chain-base hydrogen bonds favor strand and coil secondary structure types in highly specific DNA-binding proteins while multi-specific DNA-binding proteins prefer helices.

Malvaceae is a family of flowering plants containing many economically important plant species including cotton, cacao and durian. Recently, the genomes of several Malvaceae species have been decoded, and many omics data were generated for individual species. However, no integrative database of multiple species, enabling users to jointly compare and analyse relevant data, is available for Malvaceae. Thus, we developed a user-friendly database named MaGenDB (http://magen.whu.edu.cn) as a functional genomics hub for the plant community. We collected the genomes of 13 Malvaceae species, and comprehensively annotated genes from different perspectives including functional RNA/protein element, gene ontology, KEGG orthology, and gene family. We processed 374 sets of diverse omics data with the ENCODE pipelines and integrated them into a customised genome browser, and designed multiple dynamic charts to present gene/RNA/protein-level knowledge such as dynamic expression profiles and functional elements. We also implemented a smart search system for efficiently mining genes. In addition, we constructed a functional comparison system to help comparative analysis between genes on multiple features in one species or across closely related species. This database and associated tools will allow users to quickly retrieve large-scale functional information for biological discovery.

ChlamDB is a comparative genomics database containing 277 genomes covering the entire Chlamydiae phylum as well as their closest relatives belonging to the Planctomycetes-Verrucomicrobiae-Chlamydiae (PVC) superphylum. Genomes can be compared, analyzed and retrieved using accessions numbers of the most widely used databases including COG, KEGG ortholog, KEGG pathway, KEGG module, Pfam and InterPro. Gene annotations from multiple databases including UniProt (curated and automated protein annotations), KEGG (annotation of pathways), COG (orthology), TCDB (transporters), STRING (protein-protein interactions) and InterPro (domains and signatures) can be accessed in a comprehensive overview page. Candidate effectors of the Type III secretion system (T3SS) were identified using four in silico methods. The identification of orthologs among all PVC genomes allows users to perform large-scale comparative analyses and to identify orthologs of any protein in all genomes integrated in the database. Phylogenetic relationships of PVC proteins and their closest homologs in RefSeq, comparison of transmembrane domains and Pfam domains, conservation of gene neighborhood and taxonomic profiles can be visualized using dynamically generated graphs, available for download. As a central resource for researchers working on chlamydia, chlamydia-related bacteria, verrucomicrobia and planctomyces, ChlamDB facilitates the access to comprehensive annotations, integrates multiple tools for comparative genomic analyses and is freely available at https://chlamdb.ch/. Database URL: https://chlamdb.ch/.

ReMap (http://remap.univ-amu.fr) aims to provide the largest catalogs of high-quality regulatory regions resulting from a large-scale integrative analysis of hundreds of transcription factors and regulators from DNA-binding experiments in Human and Arabidopsis (Arabidopsis thaliana). In this 2020 update of ReMap we have collected, analyzed and retained after quality control 2764 new human ChIP-seq and 208 ChIP-exo datasets available from public sources. The updated human atlas totalize 5798 datasets covering a total of 1135 transcriptional regulators (TRs) with a catalog of 165 million (M) peaks. This ReMap update comes with two unique Arabidopsis regulatory catalogs. First, a catalog of 372 Arabidopsis TRs across 2.6M peaks as a result of the integration of 509 ChIP-seq and DAP-seq datasets. Second, a catalog of 33 histone modifications and variants across 4.5M peaks from the integration of 286 ChIP-seq datasets. All catalogs are made available through track hubs at Ensembl and UCSC Genome Browsers. Additionally, this update comes with a new web framework providing an interactive user-interface, including improved search features. Finally, full programmatically access to the underlying data is available using a RESTful API together with a new R Shiny interface for a TRs binding enrichment analysis tool.

The early detection of cancer holds the key to combat and control the increasing global burden of cancer morbidity and mortality. Blood-based screenings using circulating DNAs (ctDNAs), circulating RNA (ctRNAs), circulating tumor cells (CTCs) and extracellular vesicles (EVs) have shown promising prospects in the early detection of cancer. Recent high-throughput gene expression profiling of blood samples from cancer patients has provided a valuable resource for developing new biomarkers for the early detection of cancer. However, a well-organized online repository for these blood-based high-throughput gene expression data is still not available. Here, we present BBCancer (http://bbcancer.renlab.org/), a web-accessible and comprehensive open resource for providing the expression landscape of six types of RNAs, including messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), microRNAs (miRNAs), circular RNAs (circRNAs), tRNA-derived fragments (tRFRNAs) and Piwi-interacting RNAs (piRNAs) in blood samples, including plasma, CTCs and EVs, from cancer patients with various cancer types. Currently, BBCancer contains expression data of the six RNA types from 5040 normal and tumor blood samples across 15 cancer types. We believe this database will serve as a powerful platform for developing blood biomarkers.

The SIGnaling Network Open Resource 2.0 (SIGNOR 2.0) is a public repository that stores signaling information as binary causal relationships between biological entities. The captured information is represented graphically as a signed directed graph. Each signaling relationship is associated to an effect (up/down-regulation) and to the mechanism (e.g. binding, phosphorylation, transcriptional activation, etc.) causing the up/down-regulation of the target entity. Since its first release, SIGNOR has undergone a significant content increase and the number of annotated causal interactions have almost doubled. SIGNOR 2.0 now stores almost 23 000 manually-annotated causal relationships between proteins and other biologically relevant entities: chemicals, phenotypes, complexes, etc. We describe here significant changes in curation policy and a new confidence score, which is assigned to each interaction. We have also improved the compliance to the FAIR data principles by providing (i) SIGNOR stable identifiers, (ii) programmatic access through REST APIs, (iii) bioschemas and (iv) downloadable data in standard-compliant formats, such as PSI-MI CausalTAB and GMT. The data are freely accessible and downloadable at https://signor.uniroma2.it/.

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.

Plant Reactome (https://plantreactome.gramene.org) is an open-source, comparative plant pathway knowledgebase of the Gramene project. It uses Oryza sativa (rice) as a reference species for manual curation of pathways and extends pathway knowledge to another 82 plant species via gene-orthology projection using the Reactome data model and framework. It currently hosts 298 reference pathways, including metabolic and transport pathways, transcriptional networks, hormone signaling pathways, and plant developmental processes. In addition to browsing plant pathways, users can upload and analyze their omics data, such as the gene-expression data, and overlay curated or experimental gene-gene interaction data to extend pathway knowledge. The curation team actively engages researchers and students on gene and pathway curation by offering workshops and online tutorials. The Plant Reactome supports, implements and collaborates with the wider community to make data and tools related to genes, genomes, and pathways Findable, Accessible, Interoperable and Re-usable (FAIR).

The volume of biological, chemical and functional data deposited in the public domain is growing rapidly, thanks to next generation sequencing and highly-automated screening technologies. These datasets represent invaluable resources for drug discovery, particularly for less studied neglected disease pathogens. To leverage these datasets, smart and intensive data integration is required to guide computational inferences across diverse organisms. The TDR Targets chemogenomics resource integrates genomic data from human pathogens and model organisms along with information on bioactive compounds and their annotated activities. This report highlights the latest updates on the available data and functionality in TDR Targets 6. Based on chemogenomic network models providing links between inhibitors and targets, the database now incorporates network-driven target prioritizations, and novel visualizations of network subgraphs displaying chemical- and target-similarity neighborhoods along with associated target-compound bioactivity links. Available data can be browsed and queried through a new user interface, that allow users to perform prioritizations of protein targets and chemical inhibitors. As such, TDR Targets now facilitates the investigation of drug repurposing against pathogen targets, which can potentially help in identifying candidate targets for bioactive compounds with previously unknown targets. TDR Targets is available at https://tdrtargets.org.

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5' regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

Foldamers are non-natural oligomers that mimic the structural behaviour of natural peptides, proteins and nucleotides by folding into a well-defined 3D conformation in solution. Since their first description about two decades ago, numerous studies have been undertaken dealing with the design, synthesis, characterization and application of foldamers. They have huge application potential as antimicrobial, anticancer and anti-HIV agents and in materials science. Despite their importance, there is no publicly available web resource providing comprehensive information on these compounds. Here we describe FoldamerDB, an open-source, fully annotated and manually curated database of peptidic foldamers. FoldamerDB holds the information about the sequence, structure and biological activities of the foldamer entries. It contains the information on over 1319 species and 1018 activities, collected from more than 160 research papers. The web-interface is designed to be clutter-free, user-friendly and it is compatible with devices of different screen sizes. The interface allows the user to search the database, browse and filter the foldamers using multiple criteria. It also offers a detailed help page to assist new users. FoldamerDB is hoped to bridge the gap in the freely available web-based resources on foldamers and will be of interest to diverse groups of scientists from chemists to biologists. The database can be accessed at http://foldamerdb.ttk.hu/.

Pathway Commons (https://www.pathwaycommons.org) is an integrated resource of publicly available information about biological pathways including biochemical reactions, assembly of biomolecular complexes, transport and catalysis events and physical interactions involving proteins, DNA, RNA, and small molecules (e.g. metabolites and drug compounds). Data is collected from multiple providers in standard formats, including the Biological Pathway Exchange (BioPAX) language and the Proteomics Standards Initiative Molecular Interactions format, and then integrated. Pathway Commons provides biologists with (i) tools to search this comprehensive resource, (ii) a download site offering integrated bulk sets of pathway data (e.g. tables of interactions and gene sets), (iii) reusable software libraries for working with pathway information in several programming languages (Java, R, Python and Javascript) and (iv) a web service for programmatically querying the entire dataset. Visualization of pathways is supported using the Systems Biological Graphical Notation (SBGN). Pathway Commons currently contains data from 22 databases with 4794 detailed human biochemical processes (i.e. pathways) and ∼2.3 million interactions. To enhance the usability of this large resource for end-users, we develop and maintain interactive web applications and training materials that enable pathway exploration and advanced analysis.

Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction-modification (R-M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R-M system. This study might shed light on exploiting and improving CRISPR-Cas systems in other microorganisms for genome editing and metabolic engineering practices.

Here, we presented an integrative database named DrLLPS (http://llps.biocuckoo.cn/) for proteins involved in liquid-liquid phase separation (LLPS), which is a ubiquitous and crucial mechanism for spatiotemporal organization of various biochemical reactions, by creating membraneless organelles (MLOs) in eukaryotic cells. From the literature, we manually collected 150 scaffold proteins that are drivers of LLPS, 987 regulators that contribute in modulating LLPS, and 8148 potential client proteins that might be dispensable for the formation of MLOs, which were then categorized into 40 biomolecular condensates. We searched potential orthologs of these known proteins, and in total DrLLPS contained 437 887 known and potential LLPS-associated proteins in 164 eukaryotes. Furthermore, we carefully annotated LLPS-associated proteins in eight model organisms, by using the knowledge integrated from 110 widely used resources that covered 16 aspects, including protein disordered regions, domain annotations, post-translational modifications (PTMs), genetic variations, cancer mutations, molecular interactions, disease-associated information, drug-target relations, physicochemical property, protein functional annotations, protein expressions/proteomics, protein 3D structures, subcellular localizations, mRNA expressions, DNA & RNA elements, and DNA methylations. We anticipate DrLLPS can serve as a helpful resource for further analysis of LLPS.

The University of California Santa Cruz Genome Browser website (https://genome.ucsc.edu) enters its 20th year of providing high-quality genomics data visualization and genome annotations to the research community. In the past year, we have added a new option to our web BLAT tool that allows search against all genomes, a single-cell expression viewer (https://cells.ucsc.edu), a 'lollipop' plot display mode for high-density variation data, a RESTful API for data extraction and a custom-track backup feature. New datasets include Tabula Muris single-cell expression data, GeneHancer regulatory annotations, The Cancer Genome Atlas Pan-Cancer variants, Genome Reference Consortium Patch sequences, new ENCODE transcription factor binding site peaks and clusters, the Database of Genomic Variants Gold Standard Variants, Genomenon Mastermind variants and three new multi-species alignment tracks.

Endogenous retroviruses (ERVs) contribute to ∼10 percent of the mouse genome. They are often silenced in differentiated somatic cells but differentially expressed at various embryonic developmental stages. A minority of mouse embryonic stem cells (ESCs), like 2-cell cleavage embryos, highly express ERV MERVL. However, the role of ERVs and mechanism of their activation in these cells are still poorly understood. In this study, we investigated the regulation and function of the stage-specific expressed ERVs, with a particular focus on the totipotency marker MT2/MERVL. We show that the transcription factor Zscan4c functions as an activator of MT2/MERVL and 2-cell/4-cell embryo genes. Zinc finger domains of Zscan4c play an important role in this process. In addition, Zscan4c interacts with MT2 and regulates MT2-nearby 2-cell/4-cell genes through promoting enhancer activity of MT2. Furthermore, MT2 activation is accompanied by enhanced H3K4me1, H3K27ac, and H3K14ac deposition on MT2. Zscan4c also interacts with GBAF chromatin remodelling complex through SCAN domain to further activate MT2 enhancer activity. Taken together, we delineate a previously unrecognized regulatory axis that Zscan4c interacts with and activates MT2/MERVL loci and their nearby genes through epigenetic regulation.

RNA structures that impede ribosome binding or subsequent scanning of the 5'-untranslated region (5'-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5'-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5'-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such 'conditionally hyperdependent' mRNAs contain unusually long 5'-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5'-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV's largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.