Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase.

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate-distinct from that between Mcm2/5 used for origin loading-is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.

Head-on and co-directional RNA polymerase collisions orchestrate bidirectional transcription termination.

Genomic DNA is a crowded track where motor proteins frequently collide. It remains underexplored whether these collisions carry physiological function. In this work, we develop a single-molecule assay to visualize the trafficking of individual E. coli RNA polymerases (RNAPs) on DNA. Based on transcriptomic data, we hypothesize that RNAP collisions drive bidirectional transcription termination of convergent gene pairs. Single-molecule results show that the head-on collision between two converging RNAPs is necessary to prevent transcriptional readthrough but insufficient to release the RNAPs from the DNA. Remarkably, co-directional collision of a trailing RNAP into the head-on collided complex dramatically increases the termination efficiency. Furthermore, stem-loop structures formed in the nascent RNA are required for collisions to occur at well-defined positions between convergent genes. These findings suggest that physical collisions between RNAPs furnish a mechanism for transcription termination and that programmed genomic conflicts can be exploited to co-regulate the expression of multiple genes.

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1.

SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

Structure and DNA-bridging activity of the essential Rec114-Mei4 trimer interface.

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher-order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C terminus of Rec114 with the N terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide1. Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output2. However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells3 to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5' ends aligned at transcription start sites and 3' ends located 200-500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription-translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics.

A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.

High degree of coordination and division of labor among subunits in a homomeric ring ATPase.

Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.

Dynamics of Cas10 Govern Discrimination between Self and Non-self in Type III CRISPR-Cas Immunity.

Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity.

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

All-fiber hectowatt radially polarized laser system.

A high-power all-fiber radially polarized laser system is demonstrated, in which an integrated nanograting mode convertor (S-wave plate) is used for the generation of radially polarized beam. Experimentally, a 1-W radially polarized beam was used as the seed laser, whose mode purity and mode extinction ratio (MER) were 96.5% and 98.3%, respectively. A single-stage few-mode fiber amplifier was employed to boost the 1-W seed laser to an average power of 113.2 W, when the pump power was 160 W. The corresponding slope efficiency and beam quality factor (M2) were approximately 72% and 2.3%, respectively. Moreover, the mode purity and MER of the amplified radially polarized laser were measured to be 95.7% and 97%, respectively. To the best of our knowledge, this is the highest output power from an all-fiber radially polarized laser system without obvious degradations of the mode purity and MER.

Evaluating and diagnosing ecosystem health of the "three-lake" watershed in Yuxi, Yunnan, China from 2010 to 2020 by PSR-KDE.

To carry out the diagnosis and evaluation of the ecosystem health in Yuxi three-lake watershed, this paper presents the changing trend of its health state, and predicts the future development. This also provides ideas for maintaining the regional ecosystem health, and then gradually improves the ecological environment quality. Taking Fuxian Lake, Qilu Lake and Xingyun Lake (the three-lake watershed) in Yuxi City, Yunnan Province, Southwest China as the research object, a model combining pressure-state-response and kernel density estimation (PSR-KDE) adopts to diagnose and evaluate the ecosystem health of the "three lake" watershed from 2010 to 2020, and the distribution map of ecosystem health index has obtained by the evaluation indexes integration based on GIS spatial analysis. Hence, the evaluation results have visualized on the map. The results show that: The distribution of ecosystem health index in the study area was 0.1530-0.7045 in 2010, 0.2056-0.7512 in 2015, and 0.2248-0.7662 in 2020. 0.12% was in the pathological area in 2010. After 2015, the pathological condition of ecosystem health has completely solved, and the proportion of unhealthy ecosystems was 11.95% in 2010, 7.38% in 2015, and 5.97% in 2020. The ecosystem health index of the study region was 0.5523 in 2010, 0.5807 in 2015, and 0.5815 in 2020, it indicates that the ecosystem was in a sub-health state. From 2010 to 2020, the ecosystem health around Qilu Lake was the most worrying, followed by the northwest of Fuxian Lake and the northern and southern regions of Xingyun Lake. The ecosystem health of the three-lake watershed showed significant improvement from 2010 to 2020. The study ecosystem health assessment and early warning in the three-lake watershed is significant to the ecological environment protection and management of the plateau lake basin, the restoration of the territorial space ecology and the economic development of the surrounding area.

Solution-Processed Heterojunction Photodiodes Based on WSe2 Nanosheet Networks.

Solution-processed photodetectors incorporating liquid-phase-exfoliated transition metal dichalcogenide nanosheets are widely reported. However, previous studies mainly focus on the fabrication of photoconductors, rather than photodiodes which tend to be based on heterojunctions and are harder to fabricate. Especially, there are rare reports on introducing commonly used transport layers into heterojunctions based on nanosheet networks. In this study, a reliable solution-processing method is reported to fabricate heterojunction diodes with tungsten selenide (WSe2 ) nanosheets as the optical absorbing material and PEDOT: PSS and ZnO as injection/transport-layer materials. By varying the transport layer combinations, the obtained heterojunctions show rectification ratios of up to ≈104 at ±1 V in the dark, without relying on heavily doped silicon substrates. Upon illumination, the heterojunction can be operated in both photoconductor and photodiode modes and displays self-powered behaviors at zero bias.

Coherent beam combining of cylindrical vector beams for power scaling.

Coherent beam combining (CBC) of cylindrical vector beams (CVBs) based on an active phase controller is proposed and demonstrated. Experimentally, two pieces of spatially variant wave plates (S-wave plate) were employed as vector mode convertors for two individual 1064-nm fiber amplifier channels. When the system was in a closed loop, a combined output power of 10 W and a CBC efficiency of higher than 94% were achieved, for both TM01 (radially polarized) mode and TE01 (azimuthally polarized) mode cases. Moreover, the laser system showed a high vector mode purity, which was measured to be ∼97.4% (TM01) and ∼97.3% (TE01). To the best of knowledge, this could possibly be the first demonstration of CBC of CVBs, which paves the way for power scaling of CVBs.

Cooperative DNA looping by PRC2 complexes.

Polycomb repressive complex 2 (PRC2) is an essential protein complex that silences gene expression via post-translational modifications of chromatin. This paper combined homology modeling, atomistic and coarse-grained molecular dynamics simulations, and single-molecule force spectroscopy experiments to characterize both its full-length structure and PRC2-DNA interactions. Using free energy calculations with a newly parameterized protein-DNA force field, we studied a total of three potential PRC2 conformations and their impact on DNA binding and bending. Consistent with cryo-EM studies, we found that EZH2, a core subunit of PRC2, provides the primary interface for DNA binding, and its curved surface can induce DNA bending. Our simulations also predicted the C2 domain of the SUZ12 subunit to contact DNA. Multiple PRC2 complexes bind with DNA cooperatively via allosteric communication through the DNA, leading to a hairpin-like looped configuration. Single-molecule experiments support PRC2-mediated DNA looping and the role of AEBP2 in regulating such loop formation. The impact of AEBP2 can be partly understood from its association with the C2 domain, blocking C2 from DNA binding. Our study suggests that accessory proteins may regulate the genomic location of PRC2 by interfering with its DNA interactions.

Single-molecule and in silico dissection of the interaction between Polycomb repressive complex 2 and chromatin.

Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the "1-3" bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2-chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.

Mechanical regulation of the helicase activity of Zika virus NS3.

Zika virus (ZIKV) is a positive-sense single-stranded RNA virus that infects humans and can cause birth defects and neurological disorders. Its non-structural protein 3 (NS3) contains a protease domain and a helicase domain, both of which play essential roles during the viral life cycle. However, it has been shown that ZIKV NS3 has an inherently weak helicase activity, making it unable to unwind long RNA duplexes alone. How this activity is stimulated to process the viral genome and whether the two domains of NS3 are functionally coupled remain unclear. Here, we used optical tweezers to characterize the RNA-unwinding properties of ZIKV NS3-including its processivity, velocity, and step size-at the single-molecule level. We found that external forces that weaken the stability of the duplex RNA substrate significantly enhance the helicase activity of ZIKV NS3. On the other hand, we showed that the protease domain increases the binding affinity of NS3 to RNA but has only a minor effect on unwinding per se. Our findings suggest that the ZIKV NS3 helicase is activated on demand in the context of viral replication, a paradigm that may be generalizable to other flaviviruses.

Total neoadjuvant therapy for locally advanced gastric cancer and esophagogastric junction adenocarcinoma: study protocol for a prospective, multicenter, single-arm, phase II clinical trial.

BACKGROUND: Gastric cancer ranks high in terms of morbidity and mortality worldwide. Multimodal therapy is therefore essential for locally advanced gastric cancer. Recent studies have demonstrated that both perioperative chemotherapy and neoadjuvant chemoradiotherapy can improve the prognosis of patients. However, the completion rate of chemotherapy after surgery remains low, which may affect survival. Thus, identifying the best way to combine radiotherapy, chemotherapy and surgery is important. The aim of this study was to explore the toxicity and efficacy of the total neoadjuvant therapy modality for locally advanced gastric cancer. METHODS: This study will be a prospective, multicenter, single-arm, phase II clinical trial. Patients diagnosed with locally advanced (stage cT3-4 and cN positive, AJCC 8th) gastric cancer and gastroesophageal junction adenocarcinoma will be enrolled. Patients will initially receive radiotherapy (95% planned target volume: 45 Gy/25 f) and concurrent chemotherapy (S-1: 40-60 mg twice a day) followed by six cycles of consolidated chemotherapy (SOX, consisting of S-1 and oxaliplatin) and surgery. The primary objective will assess pathological complete response; the secondary objectives will include toxicities assessing surgical complications, the tumor downstaging rate and the R0 resection rate. DISCUSSION: Investigation of total neoadjuvant therapy in gastric cancer is limited. The goal of this trial is to explore the efficacy and toxicity of total neoadjuvant therapy for locally advanced gastric cancer and gastroesophageal junction adenocarcinoma. TRIAL REGISTRATION: Clinicaltrials.gov (NCT04062058, August 20, 2019).

The iRhom homology domain is indispensable for ADAM17-mediated TNFα and EGF receptor ligand release.

Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.

Using a system's equilibrium behavior to reduce its energy dissipation in nonequilibrium processes.

Cells must operate far from equilibrium, utilizing and dissipating energy continuously to maintain their organization and to avoid stasis and death. However, they must also avoid unnecessary waste of energy. Recent studies have revealed that molecular machines are extremely efficient thermodynamically compared with their macroscopic counterparts. However, the principles governing the efficient out-of-equilibrium operation of molecular machines remain a mystery. A theoretical framework has been recently formulated in which a generalized friction coefficient quantifies the energetic efficiency in nonequilibrium processes. Moreover, it posits that, to minimize energy dissipation, external control should drive the system along the reaction coordinate with a speed inversely proportional to the square root of that friction coefficient. Here, we demonstrate the utility of this theory for designing and understanding energetically efficient nonequilibrium processes through the unfolding and folding of single DNA hairpins.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.