Biology subjects

Gao, N.

Publications and source records attributed to Gao, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Paneth cell-derived Notch ligands DLL1 and DLL4 are crucial for intestinal crypt base stem cells.

Notch signaling is essential for maintaining intestinal stem cell activity and directing epithelial cell fate. Paneth cells have been proposed to be niche cells, providing Notch signal to neighboring stem cells through expression of the key Notch ligands DLL1 and DLL4. However, intestinal stem cells persist in the absence of Paneth cells. Here, we used genetic mouse models to clarify the role of Paneth cell-derived Notch ligands for stem cell function. Paneth cell-specific deletion of Dll1 and Dll4 resulted in loss of crypt base stem cells, with no effect on overall crypt cell proliferation. Organoid growth was reduced in knockout mice, suggesting reduced stem cell function. Upon irradiation injury, stem cell return and crypt regeneration were significantly impaired in the Notch ligand-deleted mice. These findings suggest that Paneth cell-derived Notch ligands DLL1 and DLL4 are crucial for crypt base stem cell maintenance and for crypt regeneration after injury.

cell biology↗

bioRxiv2026

Lamellar Normative Modelling of the Hippocampus Across the Human Lifespan

The hippocampus is a central hub of human memory and cognition and is closely associated with brain disorders. Studies have shown that it exhibits complex structural variation across the lifespan, yet the details of hippocampal morphology changes remain poorly understood. Here, we establish norms over the hippocampal geometry that resolve lamellar morphology and map lifespan trajectories across more than 27,000 individuals from 158 scanning sites. Hippocampal geometry shows spatially non-uniform developmental and ageing patterns across the lifespan, with lamellar thickness, width and length following dissociable trajectories. Across multiple brain disorders, this representation reveals localized and heterogeneous alterations beyond conventional subfield-level summaries, and uncovers a dichotomy in disease-associated patterns, with neurodegenerative conditions and schizophrenia showing predominant atrophy, whereas some other disorders exhibit focal or regionally selective hypertrophy. Transfer to a longitudinal Alzheimers Disease Neuroimaging Initiative cohort further supports out-of-sample generalization of our approach and enables individual-level tracking and conversion risk stratification. Overall, this work establishes a population-scale geometric reference for the hippocampus, extends normative brain mapping from coarse regional phenotypes to anatomically organized subcortical structure, and enables anatomically grounded characterization of disease-related alterations and individual-level deviation mapping, providing a principled basis for understanding and stratifying brain disorders across the lifespan in health and disease.

neuroscience↗

bioRxiv2026

Structural basis of receptor retro-translocation in peroxisomal protein import

Peroxisomes import all matrix proteins post-translationally from the cytosol, a process that requires recycling of cargo receptors across the peroxisomal membrane. The membrane-embedded ubiquitin ligase, composed of Pex2, Pex10, and Pex12, is central to this process, but its mechanism remains unclear. Here we determined cryo-electron microscopy structures of the Saccharomyces cerevisiae Pex2-10-12 complex in closed and open states bound to Pex8, an essential factor of previously undefined function. The structures reveal how Pex2-10-12 gates its retro-translocation pore to control receptor entry and how the closed-to-open transition repositions the Pex10 RING domain to enable receptor mono-ubiquitination. Pex8 docks onto Pex2-10-12 from the matrix and guides receptors into the pore. Functional analyses show that the receptors N-terminal segment downstream of its mono-ubiquitination site initiates a loop insertion into the pore. These findings establish how Pex2-10-12 coordinates receptor recognition, retro-translocation, and ubiquitination, providing the molecular basis for receptor recycling in peroxisomal protein import.

biochemistry↗

bioRxiv2026

Bacterial GTPases act as successive placeholders to mediate ribosome assembly and its coupling to translation initiation

Ribosome biogenesis and mRNA translation are fundamental cellular processes regulated by a diverse set of protein factors, including GTPases. In bacteria, while several GTPases are known to participate in ribosome assembly, their precise mechanisms in subunit maturation and their potential roles in translation regulation remain largely elusive. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of native pre-50S assembly intermediates and 70S translating ribosomes, isolated via epitope-tagged GTPases from engineered Escherichia coli cells at resolutions of 2.3-4.4 [A]. These structures elucidate how three GTPases, YihA, EngA and ObgE, act as successive placeholders to mediate rRNA folding and to coordinate the correct timing of the maturation of different functional blocks within the large ribosomal subunit. Furthermore, our data identify several previously unrecognized 70S translational complexes bound by the GTPases EngA and BipA--factors traditionally regarded as assembly factors, thereby uncovering their regulatory role in bridging ribosome assembly and translation initiation. Collectively, our findings delineate a GTPase-mediated surveillance system that continuously monitors the assembly of ribosomal subunits and translation adversity, thereby safeguarding protein synthesis and maintaining proteome homeostasis.

biophysics↗

bioRxiv2026

The novel variant IBDV induces inflammatory responses via VP3-mediated pyroptosis by directly targeting Caspase-9

The novel variant infectious bursal disease virus (nVarIBDV), which has been widely prevalent since 2017, threatens the poultry industry by inducing severe bursal atrophy and intense inflammatory responses. Understanding the inflammatory mechanism underlying nVarIBDV infection is critical for preventing virus-induced damage. Pyroptosis, an inflammatory type of programmed cell death, may contribute to the inflammation and bursa of Fabricius damage induced by nVarIBDV. Here, we found that the nVarIBDV infection induced severe inflammatory responses both in vivo and in vitro, which were associated with pyroptosis. Further research revealed that the viral protein VP3 drove pyroptosis induced by nVarIBDV infection by indirectly activating the Caspase-3-GSDME pathway, leading to GSDME cleavage. Mechanistically, VP3 directly interacted with and activated Caspase-9, thereby initiating Caspase-9-Caspase-3-GSDME pathway. More importantly, the Serine 33 of VP3 was identified as the key amino acid for interacting with and activating Caspase-9. Mutation of this residue significantly weakened the ability of VP3 to interact with Caspase-9 and activate the Caspase-9-mediated pyroptosis pathway, altered the binding mode of the Caspase-9-VP3 protein complex, and ultimately reduced the ability of VP3 to induce pyroptosis. In conclusion, our results elucidated a novel mechanism by which nVarIBDV infection induced inflammatory responses whereby viral protein VP3 triggered pyroptosis by targeting the Caspase-9-Caspase-3-GSDME pathway. IMPORTANCEnVarIBDV is globally prevalent and poses a significant threat to the poultry industry by causing inflammation and damage to the bursa of Fabricius. This study revealed that nVarIBDV infection induced inflammatory responses and bursa of Fabricius damage, primarily mediated by the viral protein VP3 targeting Caspase-9 to induce pyroptosis. To our knowledge, this is the first report on the interaction between inflammatory responses induced by IBDV infection and pyroptosis. In addition, serine 33 was identified as the critical amino acid required for maintaining the stability of the interaction between VP3 and Caspase-9 and activation of the Caspase-9-Caspase-3-GSDME cascade pyroptosis pathway and further inflammatory responses. Collectively, our findings not only elucidate a novel mechanism by which nVarIBDV induces pyroptosis and further leads to inflammation but also suggest that the identification of this serine site may represent a potential therapeutic target to block nVarIBDV-induced inflammation and bursa of Fabricius damage.

microbiology↗

bioRxiv2025

A VSV-vector vaccine simultaneously targeting H5N1 hemagglutinin (HA) and matrix protein 2 (M2) induces robust neutralizing and ADCC antibody responses and provides full protection against lethal H5N1 infection in a mouse model

Human (avian) influenza A viruses, especially highly pathogenic avian influenza (HPAI) viruses, pose a significant public health threat, and a multivalent vaccine is the primary prophylactic measure to control these viruses. To establish such a vaccine, we generated two multivalent vesicular stomatitis virus (VSV)-based vaccine candidates (V-EtM2e/H505 and V-EtM2e/H522) and characterized their ability to induce protective immune responses. Our results revealed that vaccine immunization in mice induced high humoral immune responses against both the HPAI hemagglutinin (HA) protein and the ectodomain of M2 (M2e) protein. Intriguingly, vaccine-immunized mouse sera exhibited highly efficient neutralizing activity against the corresponding H5 pseudovirus and mediated potent and broad antibody-dependent cellular cytotoxicity (ADCC) activity against M2e derived from human and avian influenza H5, H1, H3, and H7 viruses. Furthermore, both intranasal and intramuscular immunization provided efficient protection against HPAI H5N1 virus challenge in mice, with a 100% survival rate and a nondetectable viral load in several tissues. Notably, noninvasive mucosal (IN) delivery of V-EtM2e/H522 achieved protection equal to that of IM delivery at a 100-fold lower immunizing dose. These findings provide strong evidence for the effectiveness of a multivalent VSV-based vaccine against human (avian) influenza A viruses.

microbiology↗

bioRxiv2025

Torsins Organize CLCC1 Assembly to Safeguard ER Bilayer and Lipid Homeostasis

The TMEM41B scramblase and its regulatory partner CLCC1 initiate lipid flux by equilibrating newly-synthesized phospholipids across the endoplasmic reticulum (ER) bilayer, a fundamental process required for diverse events ranging from membrane biogenesis to bulk lipid supply. Loss of CLCC1/TMEM41B causes ER bilayer imbalance, inducing giant ER-enclosed lipid droplets (geLDs) and driving rapid progression into severe MASH. Here we identify CLCC1 as the long-missing client of the lumenal Torsin ATPases, which selectively engage oligomerized CLCC1 at sites of ER bilayer imbalance. Hepatic TorsinA inactivation triggers geLD formation amid disrupted lipoprotein biogenesis and severe MASH, closely phenocopying CLCC1/TMEM41B deficiency. Mechanistically, Torsins act as foldases that drive CLCC1 oligomerization for its recruitment to imbalanced bilayers. Remarkably, ectopic CLCC1 expression reverses cellular and systemic lipid disorders arising from hepatic TorsinA deficiency. Hence, Torsin ATPases emerge as fundamental regulators that organizes CLCC1 and the downstream TMEM41B scramblase to govern lipid partitioning and membrane homeostasis.

cell biology↗

bioRxiv2025

Creating an energy efficient central metabolism for boosting biosynthesis without compromising cell growth of yeast

Long-term natural evolution selects glycolysis as the major metabolic mode for rapid cell growth, which however lacks sufficient NADPH supply to dive the biosynthesis of reduced chemicals such as free fatty acids (FFAs). Engineering energy economical pathway for chemical overproduction always compromises cellular fitness due to the rigidity of cellular metabolism. Here, we successfully replaced glycolysis metabolism with an optimized pentose phosphate pathway (PPP) in an industrial yeast Ogataea polymorpha, for the first time, which enabled a higher energy generation efficiency than glycolysis and a balanced supply of ATP and NADPH. More importantly, we discovered a global carbon metabolism regulator CMR that drives metabolic flux toward glycolysis for energy generation, and its disruption relieved the tight regulation of metabolic flux distribution and significantly enhanced the efficiency of cellular energy generation, which significantly boosted the FFA production by 63% in a FFA overproducing chassis. The final engineered yeast produced FFAs at a titer of 41.7 g/L, the highest titer reported by microbial fermentation. Our work provides valuable insights into the metabolic regulation mechanisms and a feasible approach for constructing energy efficient metabolism for chemical overproduction.

synthetic biology↗

bioRxiv2025

Structural Role of Stomatin in Organizing Functional Membrane Microdomains

Stomatin, originally discovered in red blood cells, is a member of the SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) protein family, which has long been proposed to scaffold functional membrane microdomains (FMMs) enriched in saturated lipids such as cholesterol and sphingomyelin. Stomatin has been reported to associate with a variety of proteins involved in diverse physiological processes, including ion channel regulation, membrane fusion, mechanosensory regulation and vesicle trafficking; however, the mechanisms by which it modulates these interactions remain poorly understood. Here, we determined the cryo-electron microscopy (cryo-EM) structures of stomatin, revealing its hexadecameric assemblies. The SPFH1 domains insert into the cytosolic leaflet of membranes and, together with the N-terminal hairpin, form a potential cholesterol-binding pocket. Liposome reconstitution experiments demonstrated that stomatin self-organizes into clusters, and Spectrum and Polarization Optical Tomography (SPOT) further showed that these clusters enhance membrane lipid order, supporting the proposed role of stomatin in organizing functional membrane microdomains (FMMs). Proteomic mass spectrometry analysis identified numerous stomatin-associated cargo proteins, including solute carrier (SLC) transporters, RAB GTPases, and integrins, suggesting that stomatin regulates solute transport and contributes to vesicle trafficking and cell migration. Together, these findings elucidate the structural and functional roles of stomatin and underscore its potential as a therapeutic target for modulating cancer cell migration and metastasis.

biophysics↗

bioRxiv2025

Xenopus IgX informs engineering strategies of IgM and IgG hexamers

Polymeric immunoglobulins are essential components of the immune system in jawed vertebrates. While mammalian immunoglobulin M (IgM) typically forms a pentamer linked by the joining chain (J-chain), Xenopus laevis IgX assembles into a J-chain-independent polymer. Here, we present the cryo-electron microscopy (cryo-EM) structure of IgX, revealing its hexameric configuration. By incorporating the IgX tailpiece into human IgM, we achieved efficient IgM hexamer formation. Truncating IgMs natural tailpiece to a range of 11-16 residues also substantially enhanced hexamerization efficiency. Furthermore, introducing a shortened IgM tailpiece to IgG resulted in effective IgG hexamer formation. We further show that the engineered IgM and IgG hexamers targeting CD20 demonstrated robust complement-dependent cytotoxicity (CDC) against several B lymphoma cells. Additionally, the IgG-Fc hexamer functioned as a decoy, attenuating CDC in cell cultures. These findings deepen our understanding of polymeric immunoglobulin evolution and introduce innovative strategies for the development of IgM- and IgG-based biologics. One Sentence SummaryThe cryo-EM structure of the African clawed frog immunoglobulin IgX reveals a uniform hexameric assembly, inspiring innovative strategies for the development of therapeutic human IgM and IgG hexamers.

immunology↗

bioRxiv2025

Cryo-EM Structure of the Erlin1/2 Complex Reveals a Dynamic Scaffold for Membrane Protein Sequestration and Microdomain Assembly

The SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) family of proteins are key scaffolding components involved in the organization of functional membrane microdomains (FMMs) across various subcellular membranes, including those of the endoplasmic reticulum (ER) and mitochondria, which are characterized by a low content of saturated lipids. Among this protein family, the erlin1/2 complex is specifically located on the ER membrane. Previous studies have shown that the erlin1/2 complex plays an essential role in the ER-associated degradation (ERAD) pathway, mediating the ubiquitin-dependent degradation of various proteins such as inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are important calcium ion transporters on the ER membrane. However, the molecular mechanisms underlying erlin-mediated FMMs organization and its role in ERAD remain poorly understood. In this study, we determined the single-particle cryo-electron microscopy (cryo-EM) structure of the erlin1/2 complex under different detergent conditions. Our findings reveal that the erlin1/2 complex forms a 26-mer cage-like structure, composed of alternating erlin1 and erlin2 subunits. The erlin1/2 complex defines a nanodomain on the ER membrane, which could recruit various types of proteins to both the interior and exterior membrane regions of the cage. By caging cargo proteins, the erlin1/2 complex physically secludes them from their binding partners, leading to a potential halt in their function. Moreover, individual cages can further interact with one another, facilitating the organization of FMMs of different sizes on the ER membrane. These dynamic properties may play a general and critical role in various processes occurring on the ER, including viral replication, positioning the erlin1/2 complex as a promising new target for antiviral drug development.

biophysics↗

bioRxiv2025

The structure of phycobilisome with a bicylindrical core from the cyanobacterium Synechococcus elongatus PCC 7942

Phycobilisomes (PBSs) are the major light-harvesting complexes in the cyanobacteria and red algae and they consist of a central core and peripheral rods that are attached to the core. The PBS cores contain 2-5 allophycocyanin cylinders that are organized by ApcE. At the present, structures of PBS with tricylindrical and pentacylindrical cores have been determined while the structure of the PBS with a bicylindrical core is yet to be revealed. Here we report the cryo-EM structure of PBS with bicylindrical core from Synechococcus elongatus PCC 7942 (Synechococcus 7942) at 2.98-[A] resolution. Similar to the PBS with a tricylindrical core, six peripheral rods are attached to the core by the rod-core linker protein CpcG in the PBS of Synechococcus 7942 even though the core lacks the top AP cylinder, which is important for the attachment of peripheral rods to the tricylindrical cores. We found that the C-terminus of ApcE in the Synechococcus 7942 was involved in interacting with both CpcG and CpcB of a top peripheral rod, compensating for the absence of the top AP cylinder of the core and maintaining PBS stability. Analysis of the bilin distribution reveals that distance of excitation energy transfer from top peripheral rods to the terminal emitters is approximately 15% shorter compared to the PBS with tricylindrical cores. Although there are 30% fewer bilin chromophores in the Synechococcus 7942 PBS core compared with the tricylindrical core, the aromatic residue ring in the Synechococcus 7942 PBS core is conserved, supporting the suggestion that these aromatic residues from AP and linker proteins are critical to the energy transfer of PBS.

plant biology↗

bioRxiv2025

Structural basis for prohibitin-mediated regulation of mitochondrial m-AAA protease

Mitochondrial function critically dependents on protein quality control systems, with the m-AAA protease plays a key role at the inner mitochondrial membrane (IMM). The evolutionarily conserved prohibitins (PHBs) are essential modulators of this protease across species, yet the molecular mechanisms remain unclear. Here, we present the Cryo-EM structure of the Chaetomium thermophilum PHB (CtPHB) complex, revealing a cage-like assembly composed of 11 copies of PHB1/PHB2 heterodimers. Electron microscopic and biochemical analyses suggest that m-AAA proteases are enclosed within the PHB complex through interactions mediated by their SPFH-interacting motif (SIM) exposed in the intermembrane space. Further in situ cryo-ET directly visualizes these cage-protease assemblies in native mitochondria. Disruption of their interface leads to elevated m-AAA protease activity and diminished mitochondrial stress resistance. These data establish PHB complexes as spatial organizers that compartmentalize m-AAA proteases in membrane microdomains to fine tune proteolytic homeostasis. Our findings reveal the critical role of the PHB complex in maintaining mitochondrial proteostasis, providing a unified mechanistic model to explain and reconcile the pleiotropic, and often contradictive phenotypes of PHBs and m-AAA protease in mitochondrial physiology and various disease conditions.

molecular biology↗

bioRxiv2025

Towards machine learning fairness in classifying multicategory causes of deaths in colorectal or lung cancer patients

Classification of patient multicategory survival outcomes is important for personalized cancer treatments. Machine Learning (ML) algorithms have increasingly been used to inform healthcare decisions, but these models are vulnerable to biases in data collection and algorithm creation. ML models have previously been shown to exhibit racial bias, but their fairness towards patients from different age and sex groups have yet to be studied. Therefore, we compared the multimetric performances of 5 ML models (random forests, multinomial logistic regression, linear support vector classifier, linear discriminant analysis, and multilayer perceptron) when classifying colorectal cancer patients (n=515) of various age, sex, and racial groups using the TCGA data. All five models exhibited biases for these sociodemographic groups. We then repeated the same process on lung adenocarcinoma (n=589) to validate our findings. Surprisingly, most models tended to perform more poorly overall for the largest sociodemographic groups. Methods to optimize model performance, including testing the model on merged age, sex, or racial groups, and creating a model trained on and used for an individual or merged sociodemographic group, show potential to reduce disparities in model performance for different groups. Notably, these methods may be used to improve ML fairness while avoiding penalizing the model for exhibiting bias and thus sacrificing overall performance.

bioinformatics↗

bioRxiv2024

DnaB and DciA: Mechanisms of Helicase Loading and Translocation on ssDNA

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/622779v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b0641dorg.highwire.dtl.DTLVardef@7caf59org.highwire.dtl.DTLVardef@1dc0fccorg.highwire.dtl.DTLVardef@913ce2_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO The Vibrio cholerae (Vc) DnaB replicative helicase structure bound to single-stranded (ss) DNA is depicted in the ribbon (top left) and sphere-cylinder representation (top right). In the bottom center is a native mass spectrum showing Vc DnaB helicase loading onto single-stranded DNA (ssDNA). C_FIG Replicative helicases are assembled on chromosomes by helicase loaders before initiation of DNA replication. Here, we investigate mechanisms used by the bacterial Vibrio cholerae (Vc) DnaB replicative helicase and the DciA helicase loader. Structural analysis of the ATP{gamma}S form of the VcDnaB-ssDNA complex reveals a configuration distinct from that seen with GDP*AlF4. With ATP{gamma}S, the amino-terminal (NTD) tier, previously found as an open spiral in the GDP*AlF4 complex, adopts a closed planar arrangement. Further, the DnaB subunit at the top of the carboxy-terminal spiral (CTD) tier is displaced by [~]25 [A] between the two forms. We suggest that remodeling the NTD layer between closed planar and open spiral configurations and migration of two distinct CTDs to the top of the DnaB spiral, repeated three times, mediates hand-over-hand translocation. Biochemical analysis suggests that VcDciA leverages its Lasso domain to contact DnaB near its Docking-Linker-Helix interface. Up to three copies of VcDciA bind to VcDnaB and suppress its ATPase activity during loading onto physiological DNA substrates. Our data suggest that DciA loads DnaB onto DNA using the ring-opening mechanism.

biochemistry↗

bioRxiv2024

Revolutionizing large-scale DNA synthesis with microchip-based massive in parallel synthesis system

DNA synthesis serves as the fundamental enabling technology of engineering biology, aiming to provide DNA molecules of designed composition, length, and complexity at scale and low cost. Current high-throughput DNA synthesis technologies rely on intricate chip manufacturing and microfluidic systems to provide large-scale synthetic oligonucleotides, at the expense of low concentration and limited compatibility in the processing of longer DNA constructs assembly. Here, we report a microchip-based massive in parallel synthesis (mMPS), pioneering an "identification-sorting-synthesis-recycling" iteration mechanism to microchips for high throughput DNA synthesis. In comparison to microarray-based methods, we demonstrate that our method can increase the DNA product concentration by 4 magnitudes (to picomole-scale per sequence) and greatly simplifies the downstream processes for large-scale gene synthesis construction. By the construction of 1.97 million-diversity variant libraries that cover 1,254 human protein domains, we demonstrated the uniformity of the constructed variant libraries using mMPS-derived oligos is greatly improved, with amino acid distribution highly consistent as designed. In addition, by synthesizing 285 1kb-to-3kb genes with varying degrees of sequence complexity from previously reported strains A501 and 3DAC, potential ancestor of early archaea and bacteria, our result shows that the overall gene assembly success rate using mMPS-derived oligos is increased by 10-fold in comparison to other methods. Our mMPS technology holds the potential to close the gap between the quality and cost of writing DNA in increasing demand across many sectors of research and industrial activities.

synthetic biology↗

bioRxiv2024

Self-assembly of hybrid 3D cultures by integrating living and synthetic cells

Self-assembly, a fundamental property of living matter, drives the interconnected cellular organization of tissues. Synthetic cell models have been developed as bionic materials to mimic inherent cellular features such as self-assembly. Here, we leverage co-assembly of synthetic and natural cells to create hybrid living 3D cancer cultures. We screened synthetic cell models, including giant unilamellar vesicles, coacervates, microdroplet emulsions, proteinosomes, and colloidosomes, for their ability to form hybrid tumoroids. Our results identify the balance of inter- and extracellular adhesion and synthetic cell surface tension as key material properties driving successful co-assembly of hybrids. We further demonstrate that these synthetic cells can establish artificial tumor immune microenvironments (ART-TIMEs), mimicking immunogenic signals within tumoroids. Using the ART-TIME approach, we identify co-signaling mechanisms between PD-1 and CD2 as a driver in immune evasion of pancreatic ductal adenocarcinoma. Our findings demonstrate the 3D bottom-up self-assembly of hybrid cancer microenvironments to replace immune components with defined bionic materials, pushing the boundaries to functionally integrating living and non-living matter.

synthetic biology↗

bioRxiv2024

FACT-(H3-H4) complex stimulates Pol activity to coordinate DNA synthesis with nucleosome assembly

Deficiencies in replication-coupled (RC) nucleosome assembly often lead to reduced DNA replication rate, but the precise mechanism underlying this process remains unsolved. Here, we discovered that H3-H4, but not H2A-H2B, mediates the interaction between FACT and the primase-polymerase complex DNA Pol . This interaction stimulates the DNA polymerase activity of Pol , and is indispensable for Okazaki fragment synthesis and replication fork progression. Moreover, the Pol1-N domain of Pol provides a specific binding site for FACT and H3-H4. Furthermore, CAF-1 and Rtt106-mediated replication-coupled nucleosome assembly pathways regulate this interaction. Together, we propose that the FACT-(H3-H4)-Pol interaction acts as a "Pre-Warning System" that regulates DNA replication, ensuring proper coordination between DNA synthesis and nucleosome assembly.

molecular biology↗