The human pre-replication complex is an open complex.

In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.

Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes.

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Structure of the Spring Viraemia of Carp Virus Ribonucleoprotein Complex Reveals Its Assembly Mechanism and Application in Antiviral Drug Screening.

Spring viremia of carp virus (SVCV) is a highly pathogenic Vesiculovirus infecting the common carp, yet neither a vaccine nor effective therapies are available to treat spring viremia of carp (SVC). Like all negative-sense viruses, SVCV contains an RNA genome that is encapsidated by the nucleoprotein (N) in the form of a ribonucleoprotein (RNP) complex, which serves as the template for viral replication and transcription. Here, the three-dimensional (3D) structure of SVCV RNP was resolved through cryo-electron microscopy (cryo-EM) at a resolution of 3.7 Å. RNP assembly was stabilized by N and C loops; RNA was wrapped in the groove between the N and C lobes with 9 nt nucleotide per protomer. Combined with mutational analysis, our results elucidated the mechanism of RNP formation. The RNA binding groove of SVCV N was used as a target for drug virtual screening, and it was found suramin had a good antiviral effect. This study provided insights into RNP assembly, and anti-SVCV drug screening was performed on the basis of this structure, providing a theoretical basis and efficient drug screening method for the prevention and treatment of SVC. IMPORTANCE Aquaculture accounts for about 70% of global aquatic products, and viral diseases severely harm the development of aquaculture industry. Spring viremia of carp virus (SVCV) is the pathogen causing highly contagious spring viremia of carp (SVC) disease in cyprinids, especially common carp (Cyprinus carpio), yet neither a vaccine nor effective therapies are available to treat this disease. In this study, we have elucidated the mechanism of SVCV ribonucleoprotein complex (RNP) formation by resolving the 3D structure of SVCV RNP and screened antiviral drugs based on the structure. It is found that suramin could competitively bind to the RNA binding groove and has good antiviral effects both in vivo and in vitro. Our study provides a template for rational drug discovery efforts to treat and prevent SVCV infections.

GenTree, an integrated resource for analyzing the evolution and function of primate-specific coding genes.

The origination of new genes contributes to phenotypic evolution in humans. Two major challenges in the study of new genes are the inference of gene ages and annotation of their protein-coding potential. To tackle these challenges, we created GenTree, an integrated online database that compiles age inferences from three major methods together with functional genomic data for new genes. Genome-wide comparison of the age inference methods revealed that the synteny-based pipeline (SBP) is most suited for recently duplicated genes, whereas the protein-family-based methods are useful for ancient genes. For SBP-dated primate-specific protein-coding genes (PSGs), we performed manual evaluation based on published PSG lists and showed that SBP generated a conservative data set of PSGs by masking less reliable syntenic regions. After assessing the coding potential based on evolutionary constraint and peptide evidence from proteomic data, we curated a list of 254 PSGs with different levels of protein evidence. This list also includes 41 candidate misannotated pseudogenes that encode primate-specific short proteins. Coexpression analysis showed that PSGs are preferentially recruited into organs with rapidly evolving pathways such as spermatogenesis, immune response, mother-fetus interaction, and brain development. For brain development, primate-specific KRAB zinc-finger proteins (KZNFs) are specifically up-regulated in the mid-fetal stage, which may have contributed to the evolution of this critical stage. Altogether, hundreds of PSGs are either recruited to processes under strong selection pressure or to processes supporting an evolving novel organ.

A Rationally Designed Building Block of the Putative Magnetoreceptor MagR.

The ability of animals to perceive guidance cues from Earth's magnetic field for orientation and navigation has been supported by a wealth of behavioral experiments, yet the nature of this sensory modality remains fascinatingly unresolved and wide open for discovery. MagR has been proposed as a putative magnetoreceptor based on its intrinsic magnetism and its complexation with a previously suggested key protein in magnetosensing, cryptochrome, to form a rod-like polymer structure. Here, we report a rationally designed single-chain tetramer of MagR (SctMagR), serving as the building block of the hierarchical assembly of MagR polymer. The magnetic trapping experiment and direct magnetic measurement of SctMagR demonstrated the possibility of magnetization of nonmagnetic cells via overexpressing a single protein, which has great potential in various applications. SctMagR, as reported in this study, serves as a prototype of designed magnetic biomaterials inspired by animal magnetoreception. The features of SctMagR provide insights into the unresolved origin of the intrinsic magnetic moment, which is of considerable interest in both biology and physics. © 2022 Bioelectromagnetics Society.

Discovery of novel chemoeffectors and rational design of Escherichia coli chemoreceptor specificity.

Bacterial chemoreceptors mediate chemotactic responses to diverse stimuli. Here, by using an integrated in silico, in vitro, and in vivo approach, we screened a large compound library and found eight novel chemoeffectors for the Escherichia coli chemoreceptor Tar. Six of the eight new Tar binding compounds induce attractant responses, and two of them function as antagonists that can bind Tar without inducing downstream signaling. Comparison between the antagonist and attractant binding patterns suggests that the key interactions for chemotaxis signaling are mediated by the hydrogen bonds formed between a donor group in the attractant and the main-chain carbonyls (Y149 and/or Q152) on the α4 helix of Tar. This molecular insight for signaling is verified by converting an antagonist to an attractant when introducing an N-H group into the antagonist to restore the hydrogen bond. Similar signal triggering effect by an O-H group is also confirmed. Our study suggests that the Tar chemoeffector binding pocket may be separated into two functional regions: region I mainly contributes to binding and region II contributes to both binding and signaling. This scenario of binding and signaling suggests that Tar may be rationally designed to respond to a nonnative ligand by altering key residues in region I to strengthen binding with the novel ligand while maintaining the key interactions in region II for signaling. Following this strategy, we have successfully redesigned Tar to respond to l-arginine, a basic amino acid that does not have chemotactic effect for WT Tar, by two site-specific mutations (R69'E and R73'E).

Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family.

Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new ß-strand, nestling into two other ß-strands. Simultaneously, the apparent T(m) of the autoprocessed complex increases 20 °C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.

Determining subunit-subunit interaction from statistics of cryo-EM images: observation of nearest-neighbor coupling in a circadian clock protein complex.

Biological processes are typically actuated by dynamic multi-subunit molecular complexes. However, interactions between subunits, which govern the functions of these complexes, are hard to measure directly. Here, we develop a general approach combining cryo-EM imaging technology and statistical modeling and apply it to study the hexameric clock protein KaiC in Cyanobacteria. By clustering millions of KaiC monomer images, we identify two major conformational states of KaiC monomers. We then classify the conformational states of (>160,000) KaiC hexamers by the thirteen distinct spatial arrangements of these two subunit states in the hexamer ring. We find that distributions of the thirteen hexamer conformational patterns for two KaiC phosphorylation mutants can be fitted quantitatively by an Ising model, which reveals a significant cooperativity between neighboring subunits with phosphorylation shifting the probability of subunit conformation. Our results show that a KaiC hexamer can respond in a switch-like manner to changes in its phosphorylation level.

Synergism between CMG helicase and leading strand DNA polymerase at replication fork.

The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, one on each strand, that replicate the unwound DNA. Here, we determined a series of cryo-electron microscopy structures of a yeast replisome comprising CMG, leading-strand polymerase Polε and three accessory factors on a forked DNA. In these structures, Polε engages or disengages with the motor domains of the CMG by occupying two alternative positions, which closely correlate with the rotational movement of the single-stranded DNA around the MCM pore. During this process, the polymerase remains stably coupled to the helicase using Psf1 as a hinge. This synergism is modulated by a concerted rearrangement of ATPase sites to drive DNA translocation. The Polε-MCM coupling is not only required for CMG formation to initiate DNA replication but also facilitates the leading-strand DNA synthesis mediated by Polε. Our study elucidates a mechanism intrinsic to the replisome that coordinates the activities of CMG and Polε to negotiate any roadblocks, DNA damage, and epigenetic marks encountered during translocation along replication forks.

Menin "reads" H3K79me2 mark in a nucleosomal context.

Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.

Cryo-EM structure of TMEM63C suggests it functions as a monomer.

The TMEM63 family proteins (A, B, and C), calcium-permeable channels in animals that are preferentially activated by hypo-osmolality, have been implicated in various physiological functions. Deficiency of these channels would cause many diseases including hearing loss. However, their structures and physiological roles are not yet well understood. In this study, we determine the cryo-electron microscopy (cryo-EM) structure of the mouse TMEM63C at 3.56 Å, and revealed structural differences compared to TMEM63A, TMEM63B, and the plant orthologues OSCAs. Further structural guided mutagenesis and calcium imaging demonstrated the important roles of the coupling of TM0 and TM6 in channel activity. Additionally, we confirm that TMEM63C exists primarily as a monomer under physiological conditions, in contrast, TMEM63B is a mix of monomer and dimer in cells, suggesting that oligomerization is a regulatory mechanism for TMEM63 proteins.

Microsomal prostaglandin E synthase-1 exhibits one-third-of-the-sites reactivity.

mPGES-1 (microsomal prostaglandin E synthase-1) is a newly recognized target for the treatment of inflammatory diseases. As the terminal enzyme of the prostaglandin production pathway, mPGES-1 inhibition may have a low risk of side effects. Inhibitors of mPGES-1 have attracted considerable attention as next-generation anti-inflammatory drugs. However, as mPGES-1 is a membrane protein, its enzymatic mechanism remains to be disclosed fully. We used MD (molecular dynamics) simulations, mutation analysis, hybrid experiments and co-IP (co-immunoprecipitation) to investigate the conformation transitions of mPGES-1 during catalysis. mPGES-1 forms a homotrimer with three substrate-binding sites (pockets). In the MD simulation, only one substrate molecule could bind to one of the pockets and form the active complex, suggesting that the mPGES-1 trimer has only one pocket active at any given time. This one-third-of-the-sites reactivity enzyme mechanism was verified further by hybridization experiments and MD simulations. The results of the present study revealed for the first time a novel one-third-of-the-sites reactivity enzyme mechanism for mPGES-1, and the unique substrate-binding pocket in our model constituted an active conformation that was suitable for further enzymatic mechanism study and structural-based drug design against mPGES-1.

Pan-cancer surveys indicate cell cycle-related roles of primate-specific genes in tumors and embryonic cerebrum.

BACKGROUND: Despite having been extensively studied, it remains largely unclear why humans bear a particularly high risk of cancer. The antagonistic pleiotropy hypothesis predicts that primate-specific genes (PSGs) tend to promote tumorigenesis, while the molecular atavism hypothesis predicts that PSGs involved in tumors may represent recently derived duplicates of unicellular genes. However, these predictions have not been tested. RESULTS: By taking advantage of pan-cancer genomic data, we find the upregulation of PSGs across 13 cancer types, which is facilitated by copy-number gain and promoter hypomethylation. Meta-analyses indicate that upregulated PSGs (uPSGs) tend to promote tumorigenesis and to play cell cycle-related roles. The cell cycle-related uPSGs predominantly represent derived duplicates of unicellular genes. We prioritize 15 uPSGs and perform an in-depth analysis of one unicellular gene-derived duplicate involved in the cell cycle, DDX11. Genome-wide screening data and knockdown experiments demonstrate that DDX11 is broadly essential across cancer cell lines. Importantly, non-neutral amino acid substitution patterns and increased expression indicate that DDX11 has been under positive selection. Finally, we find that cell cycle-related uPSGs are also preferentially upregulated in the highly proliferative embryonic cerebrum. CONCLUSIONS: Consistent with the predictions of the atavism and antagonistic pleiotropy hypotheses, primate-specific genes, especially those PSGs derived from cell cycle-related genes that emerged in unicellular ancestors, contribute to the early proliferation of the human cerebrum at the cost of hitchhiking by similarly highly proliferative cancer cells.

Predicting kinetic constants of protein-protein interactions based on structural properties.

Elucidating kinetic processes of protein-protein interactions (PPI) helps to understand how basic building blocks affect overall behavior of living systems. In this study, we used structure-based properties to build predictive models for kinetic constants of PPI. A highly diverse PPI dataset, protein-protein kinetic interaction data and structures (PPKIDS), was built. PPKIDS contains 62 PPI with complex structures and kinetic constants measured experimentally. The influence of structural properties on kinetics of PPI was studied using 35 structure-based features, describing different aspects of complex structures. Linear models for the prediction of kinetic constants were built by fitting with selected subsets of structure-based features. The models gave correlation coefficients of 0.801, 0.732, and 0.770 for k(off), k(on), and K(d), respectively, in leave-one-out cross validations. The predictive models reported here use only protein complex structures as input and can be generally applied in PPI studies as well as systems biology modeling. Our study confirmed that different properties play different roles in the kinetic process of PPI. For example, k(on) was affected by overall structural features of complexes, such as the composition of secondary structures, the change of translational and rotational entropy, and the electrostatic interaction; while k(off) was determined by interfacial properties, such as number of contacted atom pairs per 100 Ų. This information provides useful hints for PPI design.

Structural study of the N-terminal domain of human MCM8/9 complex.

MCM8/9 is a complex involved in homologous recombination (HR) repair pathway. MCM8/9 dysfunction can cause genome instability and result in primary ovarian insufficiency (POI). However, the mechanism underlying these effects is largely unknown. Here, we report crystal structures of the N-terminal domains (NTDs) of MCM8 and MCM9, and build a ring-shaped NTD structure based on a 6.6 Å resolution cryoelectron microscopy map. This shows that the MCM8/9 complex forms a 3:3 heterohexamer in an alternating pattern. A positively charged DNA binding channel and a putative ssDNA exit pathway for fork DNA unwinding are revealed. Based on the atomic model, the potential effects of the clinical POI mutants are interpreted. Surprisingly, the zinc-finger motifs are found to be capable of binding an iron atom as well. Overall, our results provide a model for the formation of the MCM8/9 complex and provide a path for further studies.

Genome-wide analysis of pseudogenes reveals HBBP1's human-specific essentiality in erythropoiesis and implication in ß-thalassemia.

The human genome harbors 14,000 duplicated or retroposed pseudogenes. Given their functionality as regulatory RNAs and low conservation, we hypothesized that pseudogenes could shape human-specific phenotypes. To test this, we performed co-expression analyses and found that pseudogene exhibited tissue-specific expression, especially in the bone marrow. By incorporating genetic data, we identified a bone-marrow-specific duplicated pseudogene, HBBP1 (η-globin), which has been implicated in ß-thalassemia. Extensive functional assays demonstrated that HBBP1 is essential for erythropoiesis by binding the RNA-binding protein (RBP), HNRNPA1, to upregulate TAL1, a key regulator of erythropoiesis. The HBBP1/TAL1 interaction contributes to a milder symptom in ß-thalassemia patients. Comparative studies further indicated that the HBBP1/TAL1 interaction is human-specific. Genome-wide analyses showed that duplicated pseudogenes are often bound by RBPs and less commonly bound by microRNAs compared with retropseudogenes. Taken together, we not only demonstrate that pseudogenes can drive human evolution but also provide insights on their functional landscapes.

Visualization of Ligand-Bound Ectodomain Assembly in the Full-Length Human IGF-1 Receptor by Cryo-EM Single-Particle Analysis.

Tyrosine kinase receptor of insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) bind to hormones, such as insulin, IGF-1, and IGF-2, and transduces the signals across the cell membrane. However, the complete structure of the receptor and the signal transduction mechanism remains unclear. Here, we report the cryo-EM structure of the ligand-bound ectodomain in the full-length human IGF-1R. We reconstructed the IGF-1R/insulin complex at 4.7 Å and the IGF-1R/IGF-1 complex at 7.7 Å. Our structures reveal that only one insulin or one IGF-1 molecule binds to and activates the full-length human IGF-1R receptor.

Evolutionary transition between invertebrates and vertebrates via methylation reprogramming in embryogenesis.

Major evolutionary transitions are enigmas, and the most notable enigma is between invertebrates and vertebrates, with numerous spectacular innovations. To search for the molecular connections involved, we asked whether global epigenetic changes may offer a clue by surveying the inheritance and reprogramming of parental DNA methylation across metazoans. We focused on gametes and early embryos, where the methylomes are known to evolve divergently between fish and mammals. Here, we find that methylome reprogramming during embryogenesis occurs neither in pre-bilaterians such as cnidarians nor in protostomes such as insects, but clearly presents in deuterostomes such as echinoderms and invertebrate chordates, and then becomes more evident in vertebrates. Functional association analysis suggests that DNA methylation reprogramming is associated with development, reproduction and adaptive immunity for vertebrates, but not for invertebrates. Interestingly, the single HOX cluster of invertebrates maintains unmethylated status in all stages examined. In contrast, the multiple HOX clusters show dramatic dynamics of DNA methylation during vertebrate embryogenesis. Notably, the methylation dynamics of HOX clusters are associated with their spatiotemporal expression in mammals. Our study reveals that DNA methylation reprogramming has evolved dramatically during animal evolution, especially after the evolutionary transitions from invertebrates to vertebrates, and then to mammals.