CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.

Thermally activated history-dependent homogenization of G-quadruplexes in an ALS/FTD-associated gene.

Unraveling the inhibitory mechanism of composite allelochemicals on Microcystis aeruginosa: Integrated transcriptomic and metabolomic insights.

Although the phenotypes and functions of nonessential proteins can be studied by deletion of their coding sequences (both gene copies in diploid organisms), essential genes cannot be deleted unless loss of the encoded protein can be bypassed. Bypass is often achieved by supplementation with the product of the enzyme. However, supplementation cannot bypass loss of essential genes such as those encoding enzymes of DNA or RNA synthesis. To study proteins encoded by essential genes that cannot be bypassed, the mutations must be conditional in nature. The mutant cells must be able to grow under a permissive condition, but fail to grow under a different condition, the nonpermissive condition. Several methods have been developed to obtain conditional mutations in essential genes. Mutations that result in proteins abnormally sensitive to high temperatures are called temperature-sensitive (Ts) mutants and are a widely used type of conditional mutation. An alternative to Ts mutants is the "degron" system to target proteins for destruction by cellular proteases. Approaches to conditionally control the functions of proteins encoded by essential genes, plus the advantages and disadvantages of these and other approaches, will be considered.

Toxic effects of microplastics on extracellular polymeric substances (EPS) in estuarine microalgae under stress conditions.

Impact of extracellular polymeric substances from Skeletonema costatum on the combined toxicity of microplastics and antibiotics in estuarine environment.

Bacterial diseases pose significant threats to crop production. Natural products offer promising alternatives due to their structural diversity, low toxicity, and reduced resistance potential for controlling plant bacterial diseases. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease in cruciferous vegetables, yet effective natural products for managing black rot have not been thoroughly explored. In this study, we isolated γ-mangostin from the pericarp of mangosteen (Garcinia mangostana), which displayed significant activity against the phytopathogen Xcc with an EC50 of 6.48 ± 1.07 μg/mL. We observed that γ-mangostin exhibits strong antibacterial activity, significantly reduces the virulence of Xcc, and provides both protective and curative effects against crucifer black rot. Further mechanistic analysis reveals that γ-mangostin treatment significantly disrupts DNA synthesis, impairs swimming motility, and induces marked alterations in Xcc cell morphology. We demonstrate for the first time that γ-mangostin exerts potent antibacterial activity by compromising cellular integrity and suppressing critical virulence functions in Xcc. These findings provide a molecular foundation for advancing γ-mangostin as a sustainable biocontrol agent against Xcc for the management of crucifer black rot disease. © 2025 Society of Chemical Industry.

Iron is an essential trace element playing crucial roles in fundamental physiological processes including erythropoiesis, cell proliferation, and DNA synthesis [...]

This review examines both historical and recent evidence to clarify the current understanding of the relationship between B vitamin deficiencies and neuropathies. Vitamin B1 (thiamine) deficiency can lead to neurological disorders such as beriberi and Wernicke's encephalopathy, conditions with significant prevalence and mortality rates. Vitamin B12 (Cobalamin) is crucial for DNA synthesis, fatty acid metabolism, and myelin production, with its deficiency leading to neuropathies and cognitive disorders. Excess vitamin B6 (pyridoxine), rather than deficiency, appears to be associated with neuropathy. Vitamin B1 and B12 deficiencies are linked to classic neuropathies, while the connection between vitamin B6 deficiency and neuropathy is less clear, though excess B6 is associated with neurotoxicity. Nutritional deficiencies are less common in developed countries but remain significant in developing nations. In developed countries, factors like alcohol consumption, bariatric surgery, and metformin use are increasing these deficiencies in clinical practice.

Background/Objectives: The development of small-molecule agents that selectively target DNA replication remains a central strategy in anticancer drug discovery. In this study, we report the biological characterization of a novel 6-nitro-benzodioxin-naphthalimide (NI) derivative (compound 5a), evaluated as a potential DNA-targeted anticancer lead. Methods/Results: The antiproliferative activity of 5a was assessed in a small panel of human lung carcinoma cell models (A549, H1299) and a non-malignant control (MRC-5), revealing pronounced cytotoxic effects in tumor cells, accompanied by favorable selectivity indices. Mechanistic investigations demonstrated that treatment with 5a results in strong inhibition of DNA synthesis, as evidenced by a marked reduction in EdU incorporation and a robust induction of the DNA damage marker γH2AX. These effects were associated with cell-cycle perturbations characterized by accumulation in G1 and G2/M phases, followed by activation of apoptotic pathways. Importantly, clonogenic survival assays confirmed that even transient exposure to 5a leads to a sustained loss of proliferative capacity, indicating irreversible long-term cellular damage. These results support a replication stress-driven mechanism of action for compound 5a, consistent with interference in DNA-associated processes during S phase. Conclusions: While the precise molecular initiating event remains to be elucidated, the observed biological profile positions 5a as a promising DNA-targeted lead structure with potential for further pharmaceutical optimization. These findings provide a solid foundation for the continued development of naphthalimide-based compounds as anticancer agents within a pharmaceutically relevant framework.

The Drosophila proventriculus is a bulb-shaped structure at the juncture of the foregut and the midgut, which plays important roles in ingestion, peritrophic membrane synthesis, and the immune response to oral pathogens. A previous study identified a population of cells in the proventriculus which incorporate bromodeoxyuridine (BrdU), a marker of DNA synthesis, and proposed that these cycling cells are multipotent stem cells that replace dying cells elsewhere in the tissue. Here, we re-investigate these cycling cells and find that they do not undergo mitosis, do not generate clonal lineages, and do not proliferate in response to tissue damage, and are therefore not stem cells. Instead, we find that these cells continually endocycle throughout the fly's life, increasing their ploidy and size, while at the same time cells in this tissue are lost into the gut lumen as the fly ages. Functionally, these cells play a critical role in the synthesis of peritrophic membrane components, and we show that when theirendocycling is experimentally increased or decreased, there is a concomitant change in ploidy, tissue size, and peritrophic membrane synthesis. Further, we show that inhibition of endocycling makes flies more susceptible to orally infectious bacteria. Altogether, we show that continual endocycling of these cells is critical for maintaining tissue size and function in the face of cell loss due to aging or tissue damage.

Mycobacterium tuberculosis (M. tuberculosis) relies on the thioredoxin (Trx)–thioredoxin reductase (TrxR) system to maintain intracellular redox homeostasis and to support Trx-dependent DNA synthesis and repair, making TrxR a potential target for anti-tuberculosis therapy. Gold nanoclusters have been reported to inhibit human TrxR and suppress tumor growth, suggesting that gold-based nanomaterials can modulate TrxR activity. In this study, we report a previously uncharacterized oxidized crystal structure of M. tuberculosis TrxR containing two dimers in the asymmetric unit and use this structure to investigate inhibition by a glutathione-coated gold nanocluster (GSH-AuNC). Biolayer interferometry and enzymatic assays show that GSH-AuNC binds directly to M. tuberculosis TrxR and efficiently inhibits its catalytic activity at the purified enzyme level. Molecular dynamics simulations indicate that GSH-AuNC can occupy a surface pocket proximal to the active site, providing a plausible structural basis for enzyme engagement. AlphaFold3 modeling of the M. tuberculosis TrxR-Trx heterodimeric complex defines the interaction interface required for productive electron transfer and provides a structural hypothesis for how GSH-AuNC disrupts this process. Together, these results provide structural and mechanistic insights into the biochemical modulation of M. tuberculosis TrxR by GSH-AuNC, while the antimycobacterial activity of GSH-AuNC remains to be evaluated in future studies.

ABSTRACT Accurate replication of mitochondrial genome (mtDNA) integrity, which is essential for cellular metabolism and energy supply, relies primarily on DNA polymerase gamma (Pol γ), Twinkle helicase, and mitochondrial single-stranded DNA binding protein (mtSSB). Twinkle alone exhibits little helicase activity while reports indicate that Pol γ displays from modest to limited unwinding activity. This led us to dissect Pol γ strand displacement activity using structural, biochemical and in silico approaches. Here, we show that human Pol γ carries out robust strand displacement synthesis at physiological concentrations of divalent metal ions which reveals that distinct metal-binding sites can independently regulate DNA synthesis and unwinding activities. We further showed that Pol γ can displace RNA/DNA hybrid with comparable efficiency as DNA/DNA duplex, representing a key implication on RNA primer removal to preserve mtDNA integrity. Our cryo-electron microscopy structures of Pol γ complexed with a template containing downstream dsDNA and an incoming nucleotide revealed the structural mechanism for the strand displacement activity. We identified four conformational states that represent successive stages of DNA unwinding, accompanied by coordinated rearrangement of the downstream DNA and Pol γ elements that mediate strand displacement. This work establishes biochemical and structural mechanisms of Pol γ strand displacement activity, providing fundamental insight into human mitochondrial DNA replication and integrity. Graphical abstract

The Hepatitis B Virus (HBV) capsid assembles around an RNA pregenome which is reverse-transcribed into double-stranded DNA. It remains unclear how this DNA, which is stiff, bulky, and has significant negative charge, is accommodated within the capsid. The organization of the genome will answer this and lend insight into the viral reverse transcription reaction. We determined the structures of mature HBV and Duck HBV, finding that the DNA forms a spool that is coaxial with the capsid’s fivefold symmetry and interacts with charges on the capsid interior. We calculate that the energy of DNA bending would create a metastable capsid that just awaits a trigger for DNA release. Thus, we see how nature has created a fragile energy minimum that enables genome uncoating.

Long INterspersed Element-1 (L1) retrotransposons use activities contained within the L1 open reading frame 2-encoded protein (ORF2p) to mobilize throughout the genome via target-site primed reverse transcription (TPRT). The ORF2p endonuclease domain (EN) cleaves genomic DNA to liberate a 3’-hydroxyl (3’-OH) group that is used by the ORF2p reverse transcriptase domain (RT) to synthesize a cDNA copy of its bound L1 RNA template. L1 also can move by EN-independent retrotransposition (ENi), where a 3’-OH group at genomic DNA lesions, dysfunctional telomeres, or stalled replication forks is proposed to prime L1 reverse transcription in the absence of L1 EN cleavage. We previously reported that ribonucleoprotein (RNP) preparations from cells transfected with a human wild-type (WT) L1 or L1 EN-mutant, but not an L1 RT-mutant, can initiate reverse transcription from a DNA oligonucleotide primer/L1 RNA template complex. The WT and EN-deficient L1 RNP preparations also are associated with a nuclease activity that can process a 3’ end modification that precludes DNA synthesis from an oligonucleotide prior to priming the L1 RT reaction. Here, we purified recombinant full-length WT, L1 EN-, and L1 RT-mutant human L1 ORF2p from insect cells. We report that the WT and L1 EN-mutant, but not the L1 RT-mutant, contain an alternative endonuclease activity (alt-EN). Alt-EN activity also is detected in a bacterially expressed L1 ORF2p protein that lacks the L1 EN and ORF2p cysteine-rich domains and a thermostable group II intron-encoded protein. Processing of diverse modified primers demonstrates endonucleolytic cleavage that is eliminated by mutations in the RT active site. We propose that alt-EN is an evolutionarily conserved activity within the RT fold that promoted ENi retrotransposition of primordial retrotransposons prior to the acquisition of an EN domain.

DNA sliding clamps are essential for processive DNA synthesis in all domains of life and are loaded by ATP-dependent clamp loaders that recognize recessed 3′ ends. How clamp loaders function at nicks and small ssDNA gaps—common intermediates during DNA repair—remains incompletely understood. Here, we show that the bacterialEscherichia coliDnaX clamp loader employs a fundamentally different mechanism from its eukaryotic counterpart. Whereas eukaryotic RFC unwinds DNA at the recessed 3′ end and stabilizes the 5′-dsDNA at a dedicated shoulder site, the bacterial DnaX-complex neither unwinds DNA nor stably binds the 5′-dsDNA in vitro. Instead, cryo-EM structures reveal that the β-clamp itself contains a conserved external DNA-binding site that enables sharp bending of gapped DNA by ∼150°, promoting insertion of the 3′-dsDNA into the clamp. This DNA-bending mechanism allows efficient β-clamp loading at nicks and small gaps and reveals a distinct bacterial strategy for clamp loading. Because small DNA gaps are frequently associated with DNA damage, clamps loaded at these sites are likely important for DNA repair.In briefZheng et al. show that the bacterial clamp loader DnaX-complex uses a DNA-bending mechanism—rather than DNA unwinding—to load the β-clamp at nicks and small gaps, revealing a clamp-loading strategy distinct from eukaryotic RFC and relevant to DNA damage repair.HighlightsThe bacterial DnaX clamp loader lacks a stable shoulder DNA-binding siteUnlike eukaryotic RFC, DnaX does not unwind DNA at nicks and small gapsThe E. coli β-clamp contains a conserved external DNA-binding site absent in PCNASharp DNA bending enables β-clamp loading at nicks and small ssDNA gaps

As artificial intelligence continues to enhance biological innovation, the potential for misuse must be addressed to fully unlock the potential societal benefits. While significant work has been done to evaluate general-purpose AI and specialized biological design tools (BDTs) for biothreat creation risks, actionable steps to mitigate the risk of AI-enabled biothreat creation are underdeveloped. This paper provides policy and technology strategies collected from a diverse range of sources placed in the context of an organizing framework aligned with steps in the AI-enabled creation of a biothreat. After collating previous reports (typically on one or a small set of mitigation options) and evaluating the proposed mitigation options by projected feasibility and impact, we prioritize development of seven mitigation strategies (with a total of twelve individual mitigations): model unlearning and information removal techniques (a combination of five mitigations), classifier-based input and output filtering for BDTs, AI agents for biosecurity, safety bug bounty programs, ensuring enforcement of existing material/equipment protections, enhancing biosurveillance and bioattribution, and screening metadata/audit logs before DNA synthesis. We invite collaboration among policymakers, researchers, and technologists to refine and implement these strategies into a strong layered defense, ensuring that AI can be used safely and securely to the benefit of all.

HighlightsWhat are the main findings?TEX1 knockout parasites display severe defects in nuclear division, organelle segregation, and developmental progression during mosquito and liver stages.C-terminal HA tagging preserves TEX1 function and reveals a strong, discrete subnuclear localization pattern during liver stage development.What are the implications of the main findings?TEX1 is a critical regulator of parasite nuclear biology and liver stage schizogony, and its precise localization supports a role in chromatin- or division-associated processes.Disruption of TEX1 effectively blocks the transition from liver to blood stage, highlighting TEX1 as a promising target for liver stage and transmission-blocking interventions.Malaria remains a major global health burden, and the emergence of resistance to blood stage antimalarials underscores the need for new interventions targeting earlier stages of the parasite’s life cycle. The pre-erythrocytic liver stage represents a critical bottleneck and an attractive target for chemotherapeutic and prophylactic interventions. In this study, we functionally characterized the putative E3 ubiquitin ligase Trophozoite Exported Protein 1 (TEX1; PBANKA_0102200) in Plasmodium berghei using gene knockout, tagging, and imaging approaches across the mosquito and liver stages. TEX1 knockout parasites (PbTEX1-KO) showed impaired development during mosquito-stage transitions, with significant reductions in ookinete formation, oocyst numbers, and sporozoites reaching the salivary glands. In hepatic stages, TEX1-KO parasites displayed reduced growth, abnormal nuclear division, and impaired liver stage maturation, ultimately leading to a dramatic decline in detached cell formation and blood stage infectivity. Endogenous C-terminal tagging of TEX1 with GFP and 3×HA revealed a discrete subnuclear localization pattern, indicating a critical role in DNA synthesis and/or mitotic regulation. Our findings reveal that TEX1 is required for nuclear replication and division and successful development in both the mosquito and liver stages of Plasmodium. Given its pivotal role and nuclear localization during hepatic schizogony, TEX1 represents a promising target for the development of liver stage antimalarial interventions.

DNA Extraction Principle Inspired Instantaneous Synthesis of DNA–Gold Nanoparticle Conjugates as Sensing Probes

Terminal deoxynucleotidyl transferase (TdT) is a template-independent polymerase that catalyzes the addition of deoxynucleoside triphosphates to the 3'-terminus of a DNA strand. While TdT is a key enzyme for developing enzymatic DNA synthesis technologies, its inherent low thermal stability presents a significant limitation. This study aims to improve the thermostability of TdT from Mus musculus through a combination of site-saturation mutagenesis and rational design. Residues for saturation mutagenesis were identified using B-factor analysis and the B-FITTER program, while promising substitutions for rational design were selected using Foldit, ProteinMPNN and CARBonAra. Through several iterations of mutagenesis, we obtained two highly promising variants. The first one, dubbed A4, obtained solely through saturation mutagenesis, showed a 26-fold higher expression level than the WT protein. The second one, dubbed mutant 275, demonstrated exceptional stability, showing no significant loss of activity after 180min of incubation at 45°C - conditions under which the wild-type enzyme's half-life was less than 2min. This corresponds to a > 120-fold increase in stability. Additionally, its melting temperature (Tm) was increased by 6.5°C. Moreover, mutant 275 demonstrated a 4- to 6-fold increase in catalytic activity at 37°C. This significant enhancement in thermostability was achieved after four rounds of iterative mutagenesis and is attributed to the formation of a stabilizing salt bridge network on the protein surface, distant from the active site. The obtained thermostable TdT variants serve as robust scaffolds for further engineering to improve activity towards the 3'-reversibly blocked nucleotides required for next-generation enzymatic DNA synthesis.