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Plasmacytoid dendritic cells produce large amounts of type-I interferon (IFN-I) upon sensing nucleic acid components of pathogens by Toll-like receptors (TLR7 and TLR9). The transcription factor Spi-B has the DNA-binding Ets domain, and transactivates the Ifna4 promoter co-operatively with IFN regulatory factor-7 (IRF-7) for TLR7/TLR9-induced IFN-I production. Spi-B associates with IRF-7, and activates transcription by binding to the 5'-AGAA-3' sequence, being different from 5'-GGAA-3', known as the Ets domain recognition sequence. To understand the molecular mechanism for the co-operative transactivation of the Ifna4 promoter by Spi-B and IRF-7, we performed X-ray structural determination of the Spi-B Ets domain in complex with target DNAs, including 5'-AGAA-3' and 5'-GGAA-3' sequences. Furthermore, we conducted a modeling study of the complex of the Spi-B and IRF-7 with Ifna4 promoter DNA. X-ray structures showed that the binding of the Spi-B Ets domain induces a kink in DNA at the recognition sequence, and a more kinked DNA structure was observed in 5'-AGAA-3' than 5'-GGAA-3'. A modeling study showed that the Spi-B-induced kinked DNA structure in 5'-AGAA-3' is favorable for Spi-B and IRF-7 to approach each other for association on DNA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

In all experimental sciences, the precision and reliability of quantitative measurements are paramount. This is particularly true when examining the interactions between small molecules and biomolecules/polyelectrolytes, such as DNAs/RNAs, and yet it is overlooked in most publications of thermodynamic binding parameters. This paper presents findings from COST Action 18202 "Network for Equilibria and Chemical Thermodynamics Advanced Research," which assessed the consistency of data derived from the interactions of calf-thymus DNA (CT-DNA) with the fluorescent intercalator ethidium bromide (EB) through spectrofluorimetric titrations. We first discuss critical experimental aspects and propose a reference experimental protocol which can be used to calibrate procedures for the determination of nucleic acid binding equilibrium constants. We then fit the experimental points according to different procedures and analyse the results focusing on the statistical dispersion of the data, aiming at enlightening the strong and weak points of different fitting procedures. The implications of this work are significant, demonstrating how the statistical dispersion of experimental data can influence the interpretation of biochemical coordination mechanisms. Our study reveals that, despite rigorous protocol standardization, the determination of binding parameters remains sensitive to the choice of data fitting method, with deviations in the logarithmic stability constant (logK) values not falling below 5 % relative standard deviation (RSD), or ± 0.5 logK units for 95 % confidence. This variability evidences the critical need for standardized best practices in data treatment as well as experimental procedures. Although our study focuses on the EB/CT-DNA system through fluorescence titrations, the broader implications for other methodologies across various biochemical systems highlight the importance of this first-of-its-kind inter-laboratory comparison in advancing our understanding of biochemical coordination processes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors reports administrative support was provided by European Cooperation in Science and Technology. Eva A. Enyedy, Orsolya Domotor reports financial support was provided by National Research, Development and Innovation Office-NKFIA. E. Garcia Espana, J. Garcia reports financial support was provided by Consellería de Universidades, Ciencia y Sociedad Digital of the Generalitat Valenciana. I. Correia, N. Ribeiro, N. Basilio reports financial support was provided by Foundation for Science and Technology - Portugal. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Importin α is a crucial player in the nucleocytoplasmic transport of nuclear localization signal (NLS)-containing cargo proteins and is suggested to bind to DNA directly. We hypothesized that importin α, after binding to DNA, may move along DNA via sliding or hopping. We investigated the movement dynamics of importin αs fused to AcGFP along DNA using single-molecule fluorescence microscopy and single-tethered DNA arrays. Single-molecule data demonstrated importin α diffuses along DNA in fast and slow mobility modes. The diffusion by importin α in the fast mobility mode did not depend on salt concentration, suggesting sliding motion with continuous contact with DNA. The sliding was supported by restricted diffusion of importin α in Cas9 obstacles bound to DNA. Next, we tested whether importin α can transport a cargo molecule along DNA. Two-color imaging data established that importin α co-slides along DNA with SV40 TAg-NLS as a model cargo. We found that importin β1 together with RanGTP significantly enhanced the DNA binding of importin α and the recruitment of a model cargo TRIM28 to DNA, suggesting that importin β1/RanGTP are involved in the switching of importin α/cargo from the nuclear transport pathway to DNA sliding. Single-molecule and in vivo immunofluorescence assay demonstrates importin α assists in accumulating TRIM28 within nuclear chromatin regions. Thus, we present novel findings on the sliding dynamics and the cargo transport of importin α along DNA. The relatively faster sliding by importin α allows efficient delivery of cargo proteins to their target sites, even on long genomic DNA., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Transcription factors (TFs) such as RBPJ in Notch signaling bind to specific DNA sequences to regulate transcription. How TF-DNA binding kinetics and cofactor interactions modulate gene regulation is mostly unknown. We determine the binding kinetics, transcriptional activity, and genome-wide chromatin occupation of RBPJ and mutant variants by live-cell single-molecule tracking, reporter assays, and ChIP-Seq. Importantly, the search time of RBPJ exceeds its residence time, indicating kinetic rather than thermodynamic binding stability. Impaired RBPJ-DNA binding as in Adams-Oliver-Syndrome affect both target site association and dissociation, while impaired cofactor binding mainly alters association and unspecific binding. Moreover, our data point to the possibility that cofactor binding contributes to target site specificity. Findings for other TFs comparable to RBPJ indicate that kinetic rather than thermodynamic DNA binding stability might prevail in vivo. We propose an effective in vivo binding energy landscape of TF-DNA interactions as instructive visualization of binding kinetics and mutation-induced changes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Interactions of manganese dioxide nanoparticles (MnO 2 NPs) with vital biomolecules namely deoxyribonucleic acid (DNA) and serum albumin (BSA) have been studied in association with different surfactants by using fluorescence (steady state, synchronous and 3D), UV-visible, resonance light scattering (RLS), dynamic light scattering (DLS), and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The esterase activity of serum albumin was tested in associations with MnO 2 NPs and surfactants. The antioxidant potential of prepared NPs was also evaluated (DPPH method). Gel electrophoresis was carried out to analyze the effect of MnO 2 NPs and surfactants on DNA. Presence of CTAB, Tween 20, DTAB and Tween 80 enhanced nanoparticle-protein binding. Tween 20 based nanoparticle systems showed long-term stability and biocompatibility. The quenching of BSA fluorescence emission in presence of MnO 2 NPs alone and along with Tween 20 revealed stronger association of nanoparticles with proteins. Enhancement in the esterase activity (BSA) was observed in the presence of Tween 20. Furthermore, radical scavenging activity showed highest antioxidant potential in presence of Tween 20. The enthalpy and entropy assessment for protein-NPs association showed the predominance of Vander Waals interactions and hydrogen bonding. The synchronous fluorescence analysis highlighted the involvement of tryptophan (Trp) in the MnO 2 NPs-protein interactions. The study evaluates the influence of surfactant on the associations of MnO 2 NPs with the essential biomolecules. The findings can be crucially utilized in designing biocompatible MnO 2 formulations for long term applications.Communicated by Ramaswamy H. Sarma.

The GATA3 transcription factor is a pioneer transcription factor that is critical in the development, proliferation, and maintenance of several immune cell types. Identifying the detailed conformational dynamics and interactions of this transcription factor, as well as its clinically important population variants will allow us to unravel its mode of action. In this study, we analyze the molecular interactions of the GATA3 transcription factor bound to dsDNA as well as three clinically important population variants by atomistic molecular dynamics simulations. We identify the effect of the variants on the DNA conformational dynamics and delineate the differences compared to the wildtype transcription factor that could be related to impaired function. We highlight the structural plasticity in the binding of the GATA3 transcription factor and identify important DNA-protein contacts. Although the DNA-protein contacts are persistent and appear to be stable, they exhibit nanosecond timescale fluctuations and several binding/unbinding events. Further, we identify differential DNA binding in the three variants and show that the N-terminal binding is reduced in two of the variants. Our results indicate that reduced minor groove width and DNA diameter are important hallmarks for the binding of GATA3. Our work is an important step towards understanding the functional dynamics of the GATA3 protein and its clinically significant population variants., (© 2024 Wiley Periodicals LLC.)

Despite its limitations, restriction enzyme (RE)-mediated cleavage remains the prevalent method for generating sticky ends in DNA assembly. Here, we present RNase HII Fusion (RH2Fusion), a robust system for user-defined sticky ends, enabling scarless assembly of multiple DNA fragments alongside simultaneous site-directed mutagenesis (SDM) at multiple sites. In bacterial cells, DNA fragments with ribonucleotide modifications are expected to form complementary 3' overhangs after RNase HII treatment, followed by annealing and recombination via the bacterial self-repair system. In vitro, RNase HII-mediated cleavage produces similar overhangs, which are subsequently processed and ligated by YgdG and T4 DNA ligase, enabling efficient DNA assembly. We report for the first time that Escherichia coli Exonuclease IX (YgdG) possesses ribonuclease-specific cleavage activity, selectively cleaving ribonucleotides without cleaving deoxyribonucleotides. Through the fusion of RNase HII and YgdG, novel constructs RNase RY (RNase HII-YgdG) and RNase YR (YgdG-RNase HII) are generated, each showcasing dual enzyme functionality. In conclusion, RH2Fusion offers a rapid, effective, and versatile alternative for DNA assembly, empowering researchers across diverse fields like synthetic biology and genetic engineering. This transformative tool is poised to significantly enhance the capabilities of DNA manipulation and advance molecular biology research., Competing Interests: Declaration of competing interest The authors declare competing financial interests: Y.Z. and B.L. have filed patent applications on this work through Xiamen University., (Copyright © 2024 Elsevier B.V. All rights reserved.)

1,2,3-Triazole compounds ( 1a-3a ) and their oxime derivatives ( 1b-3b ) were synthesized. The structures of these synthesized compounds were characterized using common spectroscopic methods. Crystal structures of the compounds 3, 2b and 3b were determined by single crystal X-ray diffraction studies. The acetylcholinesteras (AChE) and butyrylcholinesterase (BChE) cholinesterase inhibitor (ChEI) and DNA/calf serum albumin (BSA) binding properties of the compounds were examined. DNA binding studies have shown that compounds interact with DNA through 1,2,3-triazole and oxime groups. When the binding constant K b values were compared, it was revealed that compound 3b ( K b  = 4.6 × 10 5 M -1 ) with oxime in its structure binds more strongly than the others. In addition, in vitro BSA binding studies showed that compounds 1b and 3b exhibited higher binding affinity. These results confirm that the quenching is due to the formation of a compound resulting from the static quenching mechanism, rather than being initiated by a dynamic mechanism. Likewise, when the enzyme activity of the compounds was examined, the compounds exhibited high inhibitory activity against AChE. The highest activity was observed for compounds 2b and 3b (8.6 ± 0.05 and 4.8 ± 0.052 µM). It was observed that the compounds were not selective with respect to BChE. Communicated by Ramaswamy H. Sarma.

Considering p53's pivotal role as a tumor suppressor protein, proactive identification and characterization of potentially harmful p53 mutations are crucial before they appear in the population. To address this, four computational prediction tools-SIFT, Polyphen-2, PhD-SNP, and MutPred2-utilizing sequence-based and machine-learning algorithms, were employed to identify potentially deleterious p53 nsSNPs (nonsynonymous single nucleotide polymorphisms) that may impact p53 structure, dynamics, and binding with DNA. These computational methods identified three variants, namely, C141Y, C238S, and L265P, as detrimental to p53 stability. Furthermore, molecular dynamics (MD) simulations revealed that all three variants exhibited heightened structural flexibility compared to the native protein, especially the C141Y and L265P mutations. Consequently, due to the altered structure of mutant p53, the DNA-binding affinity of all three variants decreased by approximately 1.8 to 9.7 times compared to wild-type p53 binding with DNA (14 μM). Notably, the L265P mutation exhibited an approximately ten-fold greater reduction in binding affinity. Consequently, the presence of the L265P mutation in p53 could pose a substantial risk to humans. Given that p53 regulates abnormal tumor growth, this research carries significant implications for surveillance efforts and the development of anticancer therapies., (© 2024 Wiley Periodicals LLC.)

The P53 protein, a cancer-associated transcriptional factor and tumor suppressor, houses a Zn 2+ ion in its DNA-binding domain (DBD), essential for sequence-specific DNA binding. However, common mutations at position 273, specifically from Arginine to Histidine and Cysteine, lead to a loss of function as a tumor suppressor, also called DNA contact mutations. The mutant (MT) P53 structure cannot stabilize DNA due to inadequate interaction. To investigate the conformational changes, we performed a comparative molecular dynamic simulation (MDS) to study the effect of the P53-Wildtype (P53-WT) and the DNA contact mutations (R273H and R273C) on the DBD. Our research indicated that the DNA binding bases lose Hydrogen bonds (H bonds) when mutated to P53-R273H and P53-R273C during the simulation. We employed tools, such as PDIviz to highlight the contacts with DNA bases and backbone, major and minor grooves, and various pharmacophore forms of atoms. The contact maps for R273H and R273C were generated using the COZOID tool, which displayed changes in the frequency of the amino acids and DNA bases interaction in the DNA binding domain. These residues have diminished interactions, and the zinc-binding domain shows significant movements by Zn 2+ ion binding to the phosphate group of the DNA, moving away from its binding sites. In conclusion, our research suggests that R273H and R273C each have unique stability and self-assembly properties. This understanding might assist researchers in better comprehending the function of the p53 protein and its importance in cancer.Communicated by Ramaswamy H. Sarma.

The study delves into the binding properties of acridine-9-amine and its selected, mainly N-substituted derivatives (A9As), with calf thymus deoxyribonucleic acid (CT-DNA). This investigation, conducted using UV-Vis spectrophotometry, steady-state fluorescence spectroscopy and isothermal titration calorimetry, provides insights into the relationship between their structure and activity. The absorption spectra of the A9As exhibited a slight red shift and significant hypochromic effects, while the fluorescence emission intensities decreased in the presence of CT-DNA. These results suggest that all fluorescent substrates intercalate into the double helix of native DNA to varying degrees. The binding constants for the A9As/CT-DNA complexes (log(K A ) were determined using various techniques in the range from 2.59 to 5.50). The thermodynamic parameters of A9As binding to DNA were obtained from ITC measurements (ΔG from - 7.51 to - 6.75 kcal·mol -1 , ΔH from - 11.58 to - 3.83 kcal·mol -1 , and TΔS from - 4.83 to 3.68 kcal·mol -1 ) and indicated that the formation of all the investigated A9As-DNA complexes is an enthalpy-driven process. The study also discusses the influence of the emitters' structure and electronic properties of substituents on intercalation efficiency. This knowledge serves as a guide for further research and offers directions for functionalising new acridines as potential reagents. It also provides the latest information on the ability of intercalation to DNA, which can be instrumental in studies on the mechanism of binding small aromatic molecules to DNA and can potentially contribute to new anticancer drug designs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein post-translational modification (PTM) serves as an important mechanism for regulating protein function. Accurate assay of PTM stoichiometry, or PTM occupancy, which refers to the proportion of proteins that contain specific modifications, is important for understanding the function of PTMs. We previously developed a novel chemoproteomic strategy "STO-MS" to quantify the PTM stoichiometry in complex biological samples, which employs a resolvable polymer mass tag to differentiate modified proteins and utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques to measure PTM stoichiometry. However, the resolution of STO-MS is constrained by the relatively low molecular weight of the mass tag, and the incorporation of isotopic labels not only complicates the sample preparation but also restricts the measurement throughput. To address these challenges, we herein developed "STO-MS+", an enhanced workflow, that incorporates an optimized DNA mass tag and employs a label-free quantitative data analysis approach. We applied STO-MS+ to measure stoichiometry of three distinct PTMs, including endogenous carbonylation induced by arachidonic acid (AA), itaconation, and endogenous O-GlcNAcylation. Our work marks a notable improvement in chemoproteomic methodologies for quantifying post-translational modifications and provides a powerful analytical tool for PTM research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cyclic GMP-AMP synthase (cGAS) is a major cytosolic DNA sensor that plays a significant role in innate immunity. Upon binding to double stranded DNA (dsDNA), cGAS utilizes GTP and ATP to synthesize the second messenger cyclic GMP-AMP (cGAMP). The cGAMP then binds to the adapter protein stimulator of interferon genes (STING) in the endoplasmic reticulum, resulting in the activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent induction of type I interferon. An important question is how cGAS distinguishes between self and non-self DNA. While cGAS binds to the phosphate backbone of DNA without discrimination, its activation is influenced by physical features such as DNA length, inter-DNA distance, and mechanical flexibility. This suggests that the recognition of DNA by cGAS may depend on these physical features. In this article we summarize the recent progress in research on cGAS-STING pathway involved in antiviral defense, cellular senescence and anti-tumor response, and focus on DNA recognition mechanisms based on the physical features., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

The interaction of drugs with DNA is crucial for understanding their mechanism of action, particularly in the context of gene expression regulation. Erdafitinib (EDB), a pan-FGFR (fibroblast growth factor receptor) inhibitor approved by the FDA, is a potent anticancer agent used primarily in the treatment of urothelial carcinoma. In this study, the binding interaction between EDB and calf thymus DNA (ctDNA) was assessed using molecular docking, UV-absorption spectroscopy, fluorescence spectroscopy, and circular dichroism (CD) spectroscopy. The absorption spectra indicated a hypochromic effect when EDB was combined with ctDNA. The binding constant (K a ) of EDB-ctDNA complex was calculated as 7.84 × 10 3  M -1 , corresponds to a free energy change (ΔG) value of approximately -5.06 kcal/mol, indicating a moderate binding affinity. Fluorometric analysis revealed a static binding mechanism in the ground state, with a bimolecular enhancement constant (K B ) of 7.56 × 10 11  M -1 . Displacement experiments demonstrated that EDB preferentially binds to the minor groove of ctDNA, with a Ksv value of 5.14 × 10 4  M -1 . Further, KI quenching and CD spectroscopy confirmed the minor groove binding mode, which was associated with a decrease in the T m from 68.28 °C to 65.84 °C, reflecting a destabilizing effect on DNA helix. Molecular docking supported these findings, showing that EDB exhibits a strong affinity for the minor groove of ctDNA and hydrogen bonding and Vander Waal interactions are the major forces involved in the binding. These results suggest that EDB primarily binds to the minor groove of ctDNA, which may play a role in its anticancer activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Nucleosomes, the building block of chromatin, are responsible for regulating access to the DNA sequence. This control is critical for essential cellular processes, including transcription and DNA replication and repair. Studying chromatin can be challenging both in vitro and in vivo, leading many to use a mono-nucleosome system to answer fundamental questions relating to chromatin regulators and binding partners. However, the mono-nucleosome fails to capture essential features of the chromatin structure, such as higher-order chromatin folding, local nucleosome-nucleosome interactions, and linker DNA trajectory and flexibility. We briefly review significant discoveries enabled by the mono-nucleosome and emphasize the need to go beyond this model system in vitro. Di-, tri-, and tetra-nucleosome arrays can answer important questions about chromatin folding, function, and dynamics. These multi-nucleosome arrays have highlighted the effects of varying linker DNA lengths, binding partners, and histone post-translational modifications in a more chromatin-like environment. We identify various chromatin regulatory mechanisms yet to be explored with multi-nucleosome arrays. Combined with in-solution biophysical techniques, studies of minimal multi-nucleosome chromatin models are feasible., (© 2025 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used for this application, but solid-state nanopore approaches have lagged. This is due to the necessity of using higher salt conditions (e.g., 4 M LiCl) to improve the signal-to-noise ratio which completely abolish the activities of many biochemical reactions. We pioneered a polymer electrolyte solid-state nanopore approach that maintains a high signal-to-noise ratio even at a physiologically relevant salt concentration. Here, we report the monitoring of the restriction enzyme SwaI and CRISPR-Cas9 endonuclease activities under physiological salt conditions and in real time. We investigated the dsDNA cleavage activity of these enzymes in a range of digestion buffers and elucidated the off-target activity of CRISPR-Cas9 ribonucleoprotein endonuclease in the presence of single base pair mismatches. This approach enables the application of solid-state nanopores for the dynamic monitoring of biochemical reactions under physiological salt conditions.

The WRN protein is vital for managing perturbed replication forks. Replication Protein A strongly enhances WRN helicase activity in specific in vitro assays. However, the in vivo significance of RPA binding to WRN has largely remained unexplored. We identify several conserved phosphorylation sites in the acidic domain of WRN targeted by Casein Kinase 2. These phosphorylation sites are crucial for WRN-RPA interaction. Using an unphosphorylable WRN mutant, which lacks the ability to bind RPA, we determine that the WRN-RPA complex plays a critical role in fork recovery after replication stress countering the persistence of G4 structures after fork stalling. However, the interaction between WRN and RPA is not necessary for the processing of replication forks when they collapse. The absence of WRN-RPA binding hampers fork recovery, causing single-strand DNA gaps, enlarged by MRE11, and triggering MUS81-dependent double-strand breaks, which require repair by RAD51 to prevent excessive DNA damage., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Enzymes encapsulated in extracellular vesicles (EVs) hold promise as biomarkers for early cancer diagnosis. However, precise measurement of their catalytic activities within EVs remains a notable challenge. Here, we report an enzymatically triggered spherical DNA nanomotor (EDM) that enables one-pot, cascaded, and highly sensitive analysis of the activity of EV-associated or free apurinic/apyrimidinic endonuclease 1 (APE1, a key enzyme in base excision repair) across various biological samples. The EDM capitalizes on APE1-triggered activation of DNAzyme (Dz) and its autonomous cleavage of substrates to achieve nonlinear signal amplification. Using EDM, we demonstrate a strong correlation between APE1 activity in EVs and that of their parental cancer cells. Additionally, EV APE1 mirrors the fluctuation of cellular APE1 activity in response to chemotherapy-induced DNA damage. In a pilot clinical study (n=63), the EDM-based assay reveals that more than 80 % of active APE1 in serum samples is EV-encapsulated. Notably, EV APE1 can differentiate early prostate cancer (PCa) patients from healthy donors (HDs) with an overall accuracy of 92 %, outperforming free APE1 in sera. We anticipate that EDM will become a versatile tool for quantifying EV-associated enzymes., (© 2024 Wiley-VCH GmbH.)

To tolerate oxidative stress, cells enable DNA repair responses often sensitive to poly(ADP-ribose) (PAR) polymerase 1 and 2 (PARP1/2) inhibition-an intervention effective against cancers lacking BRCA1/2. Here, we demonstrate that mutating the CHD6 chromatin remodeler sensitizes cells to PARP1/2 inhibitors in a manner distinct from BRCA1, and that CHD6 recruitment to DNA damage requires cooperation between PAR- and DNA-binding domains essential for nucleosome sliding activity. CHD6 displays direct PAR-binding, interacts with PARP-1 and other PAR-associated proteins, and combined DNA- and PAR-binding loss eliminates CHD6 relocalization to DNA damage. While CHD6 loss does not impair RAD51 foci formation or DNA double-strand break repair, it causes sensitivity to replication stress, and PARP1/2-trapping or Pol ζ inhibitor-induced γH2AX foci accumulation in S-phase. DNA repair pathway screening reveals that CHD6 loss elicits insufficiency in apurinic-apyrimidinic endonuclease (APEX1) activity and genomic abasic site accumulation. We reveal APEX1-linked roles for CHD6 important for understanding PARP1/2-trapping inhibitor sensitivity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The cytotoxicity of four rhenium compounds: fac-[ReO 3 (impy)CH 3 ] (1) (impy = 2-(1H-imidazole-2-yl)pyridine), fac-[Re(CO) 3 (bzimpy)Cl] (2) (bzimpy = 2-(2-pyridyl)benzimidazole), fac-[Re(CO) 3 (bibzimpy)Cl] (3) (bibzimpy = 2,6-bis(2-benzimidazolyl)pyridine) and fac-[Re(CO) 3 (impy)Cl] (4) was assessed against cancer cell lines, namely, the cervical hormone-responsive HeLa and the triple-negative breast cancer (TNBC) HCC70 lines versus a non-tumorigenic control breast epithelial cell line, MCF12A. A rare facial trioxorhenium(VII) compound 1 was characterized via various physicochemical techniques. The rhenium compounds 1-4 were, in general, more cytotoxic to HeLa cells, compared to the TNBC HCC70 line, displaying half maximal inhibitory concentration (IC 50 ) values in the micromolar range, however, the compounds were not convincingly selective for cancer cells over non-cancerous cells. In particular, compound 4 was highly cytotoxic towards HCC70, HeLa, and MCF12A cells, displaying low micromolar toxicity with IC 50 values of 6.57 ± 1.11 μM, 8.88 ± 1.07 and 9.41 ± 1.04 μM in these three cell lines, respectively and was selected for further study as it displayed the greatest cytotoxicity against the highly treatment-resistant HCC70 TNBC cell line. Compound 4 was able to both bind to genomic DNA and act as an intercalator of CT-DNA, however, this did not lead to DNA damage as assessed by a comet assay. In addition, Compound 4 displayed a long-term dose-dependent effect on colony formation and long-term survival as a proxy of in vivo toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Large vertebrate genomes duplicate by activating tens of thousands of DNA replication origins, irregularly spaced along the genome. The spatial and temporal regulation of the replication process is not yet fully understood. To investigate the DNA replication dynamics, we developed a methodology called RepliCorr, which uses the spatial correlation between replication patterns observed on stretched single-molecule DNA obtained by either DNA combing or high-throughput optical mapping. The analysis revealed two independent spatiotemporal processes that regulate the replication dynamics in the Xenopus model system. These mechanisms are referred to as a fast and a slow replication mode, differing by their opposite replication fork speed and rate of origin firing. We found that Polo-like kinase 1 (Plk1) depletion abolished the spatial separation of these two replication modes. In contrast, neither replication checkpoint inhibition nor Rap1-interacting factor (Rif1) depletion affected the distribution of these replication patterns. These results suggest that Plk1 plays an essential role in the local coordination of the spatial replication program and the initiation-elongation coupling along the chromosomes in Xenopus, ensuring the timely completion of the S phase., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing its inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3' end modifications and de-modifications, which can regulate LbCas12a's cis- and trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA modifications at the DR 3' end on LbCas12a's functionality. We find that the temporary inhibitory function of Cas12a can be reactivated by DR 3' end modification corresponding substances, such as alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), DNA exonucleases, ultraviolet radiation, and DNA glycosylases, which greatly expand the scope of application of Cas12a. Clinical applications demonstrated promising results in ALP, AFP, and trace Epstein-Barr virus detection compared to gold standard methods. Our research provides valuable insights into regulating LbCas12a activity through direct modification of DR and significantly expands its potential clinical detection targets, paving the way for future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Growth in the development of engineered polymerases for synthetic biology has led to renewed interest in assays that can measure the fidelity of polymerases that are capable of synthesizing artificial genetic polymers (XNAs). Conventional approaches require purifying the XNA intermediate of a replication cycle (DNA → XNA → DNA) by denaturing polyacrylamide gel electrophoresis, which is a slow, costly, and inefficient process that requires a large-scale transcription reaction and careful extraction of the XNA strand from the gel slice. In an effort to streamline the assay, we developed a purification-free approach in which the XNA transcription and reverse transcription steps occur inside the matrix of a hydrogel-coated magnetic particle. Accordingly, a DNA primer cross-linked throughout the gel matrix is annealed to a template of defined sequence and extended with XNA. Following removal of the DNA template, the XNA product strand is copied back into DNA, recovered, amplified, cloned, and sequenced. Performing the replication cycle in the hydrogel format drastically reduces the time and reaction scales required to measure the fidelity of an XNA polymerase, making it easier to evaluate the properties of a range of candidate XNA polymerases., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions necessary for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin of replication, catalyzing the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. We report the cryo-electron microscopy structures of Rep68 bound to the adeno-associated virus integration site 1 in different nucleotide-bound states. In the nucleotide-free state, Rep68 forms a heptameric complex around DNA, with three origin-binding domains (OBDs) bound to the Rep-binding element sequence, while three remaining OBDs form transient dimers with them. The AAA+ domains form an open ring without interactions between subunits and DNA. We hypothesize that the heptameric structure is crucial for loading Rep68 onto double-stranded DNA. The ATPγS complex shows that only three subunits associate with the nucleotide, leading to a conformational change that promotes the formation of both intersubunit and DNA interactions. Moreover, three phenylalanine residues in the AAA+ domain induce a steric distortion in the DNA. Our study provides insights into how an SF3 helicase assembles on DNA and provides insights into the DNA melting process., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Prokaryotic Argonaute proteins (pAgos) defend the host against invading nucleic acids, including plasmids and viruses. Short pAgo systems confer immunity by inducing cell death upon detecting invading nucleic acids. However, the activation mechanism of the SiAgo system, comprising a short pAgo from the archaeon Sulfolobus islandicus and its associated proteins SiAga1 and SiAga2, remains largely unknown. Here, we determined the cryo-electron microscopy structures of the SiAgo-Aga1 apo complex and the RNA-DNA-bound SiAgo-Aga1 complex at resolutions of 2.7 and 3.0 Å, respectively. Our results revealed that a positively charged pocket is generated from the interaction between SiAgo and SiAga1, exhibiting an architecture similar to APAZ-pAgo of short pAgo systems and accommodating the nucleic acids. Further investigation elucidated the conserved mechanism of nucleic acid recognition by SiAgo-Aga1. Both the SiAgo-Aga1 interaction and nucleic acid recognition by the complex are essential for antiviral defense. Biochemical and structural analyses demonstrated that SiAgo-Aga1 undergoes extensive conformational changes upon binding to the RNA-DNA duplex, thereby licensing its interaction with the effector SiAga2 to trigger the immune response. Overall, our findings highlight the evolutionary conservation of Agos across phylogenetic clades and provide structural insights into the activation mechanism of the SiAgo system., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The design of functional artificial cells involves compartmentalizing biochemical processes to mimic cellular organization. To emulate the complex chemical systems in biological cells, it is necessary to incorporate an increasing number of cellular functions into single compartments. Artificial organelles that spatially segregate reactions inside artificial cells will be beneficial in this context by rectifying biochemical pathways. Here, we develop artificial cells with all-aqueous droplet-in-droplet structures that separate transcription and translation processes like the nucleus and cytosol in eukaryotic cells. This architecture uses protein-based inner droplets and aqueous two-phase outer compartments, stabilized by colloidal emulsifiers. The inner droplet is designed to enrich DNA and RNA polymerase for transcription, coupled to translation at the outer droplet via mRNA-mediated cascade reactions. We show that these processes proceed independently within each compartment, maintaining genotype-phenotype correspondence. This approach provides a practical tool for exploring complex systems of artificial organelles within large ensembles of artificial cells., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small DNA-binding protein that specifically targets AT-rich DNA sequences. Structurally, HMGA2 is an intrinsically disordered protein (IDP), comprising three positively charged 'AT-hooks' and a negatively charged C-terminus. HMGA2 can form homodimers through electrostatic interactions between its 'AT-hooks' and C-terminus. This suggests that the negatively charged C-terminus may inhibit DNA binding by interacting with the positively charged 'AT-hooks.' In this paper, we demonstrate that the C-terminus significantly influences HMGA2's DNA-binding properties. For example, the C-terminal deletion mutant HMGA2Δ95-108 binds more tightly to the AT-rich DNA oligomer FL814 than wild-type HMGA2. Additionally, a synthetic peptide derived from the C-terminus (the C-terminal motif peptide or CTMP) strongly inhibits HMGA2's binding to FL814, likely by interacting with the 'AT-hooks,' as shown by various biochemical and biophysical assays. Molecular modeling demonstrates that electrostatic interactions and hydrogen bonding are the primary forces driving CTMP's binding to the 'AT-hooks.' Intriguingly, we found that hydration does not play a role in HMGA2-DNA binding. These results suggest that the highly negatively charged C-terminus of HMGA2 plays a critical role in regulating its DNA-binding capacity through autoinhibition, likely facilitating the target search process for AT-rich DNA sequences., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Maintaining genome integrity is an essential and challenging process. RAD51 recombinase, the central component of several crucial processes in repairing DNA and protecting genome integrity, forms filaments on DNA, which are tightly regulated. One of these RAD51 regulators is FIGNL1 (fidgetin-like 1), which prevents RAD51 genotoxic chromatin association in normal cells and persistent RAD51 foci upon DNA damage. The cryogenic electron microscopy-imaged structure of FIGNL1 in complex with RAD51 reveals that FIGNL1 forms a nonplanar hexamer and encloses RAD51 N terminus in the FIGNL1 hexamer pore. Mutations in pore loop or catalytic residues of FIGNL1 render it defective in filament disassembly and are lethal in mouse embryonic stem cells. Our study reveals a distinct mechanism for removing RAD51 from bound substrates and provides the molecular basis for FIGNL1 in maintaining genome stability.

The emergence of nanopores in two-dimensional (2D) nanomaterials offers an attractive solid-state platform for high-throughput and low-cost DNA sequencing. However, several challenges remain to be addressed before their wide application, including the too-fast DNA translocation speed (compared to state-of-the-art single nucleoside detection techniques) and too large noise/signal ratios due to DNA fluctuations inside the nanopores. Here, we use molecular dynamics (MD) simulations to demonstrate the feasibility of utilizing RNA-DNA interactions in modulating DNA translocations in 2D MoS 2 nanopores. By constructing a transmembrane-RNA-oligonucleotide-decorated nanopore (TOD nanopore), we find that the translocation speed of DNA can be significantly slowed in a sequence-dependent manner, with up to 160-fold deceleration compared with the naked control. The strong interactions between the translocating DNA and the first and second guanines of transmembrane RNAs are thought to play a key role in regulating the translocation process. Moreover, the observed suppression of base conformational fluctuations within the TOD nanopore can further improve the single nucleotide detecting resolution. Therefore, our investigations demonstrate that the proposed TOD nanopore can be a potential candidate for enhanced DNA sequencing with solid-state nanopores.

This study extends previous research, particularly focusing on patented scientific objects No. ID: PL 240 353 B1, investigating the physicochemical properties of the methyl 3-azido- and 3-amino-2,3-dideoxysaccharides with a nucleoside scaffold similar to 3'-azidothymidine (AZT). The study utilizes multiwavelength spectrophotometric and potentiometric methods to evaluate the ionization of the saccharide units in aqueous solutions. p K a values, obtained from two independent methods, reveal significant sugar ionization effects on UV spectra with varying pH levels. Stability constants for divalent metal ion complexes (Cu 2+ and Ni 2+ ) with the saccharide isomers indicate that complex stoichiometries and stabilities are highly dependent on the configuration of sugar ring substituents. Spectrophotometric results show a descending order of CT -DNA-binding affinity: BRNH 2 OMe > BRN 3 OMe > ARN 3 OMe > ARNH 2 OMe , suggesting varied interaction strengths. Molecular docking of models of synthesized O -glycosides confirmed their potential as reverse transcriptase inhibitors. Among the derivatives tested, the compound BRN 3 OMe displays the highest interaction with the enzyme active site residues and DNA, suggesting it may possess the greatest efficacy. Our reported results highlight the promising inhibitory properties of novel O -glycosides against HIV reverse transcriptase, supporting their potential development as antiviral agents.

DNA-joining by ligase and polymerase enzymes has provided the foundational tools for generating recombinant DNA and enabled the assembly of gene and genome-sized synthetic products. Xenobiotic nucleic acid (XNA) analogues of DNA and RNA with alternatives to the canonical bases, so-called 'unnatural' nucleobase pairs (UBP-XNAs), represent the next frontier of nucleic acid technologies, with applications as novel therapeutics and in engineering semi-synthetic biological organisms. To realise the full potential of UBP-XNAs, researchers require a suite of compatible enzymes for processing nucleic acids on a par with those already available for manipulating canonical DNA. In particular, enzymes able to join UBP-XNA will be essential for generating large assemblies and also hold promise in the synthesis of single-stranded oligonucleotides. Here, we review recent and emerging advances in the DNA-joining enzymes, DNA polymerases and DNA ligases, and describe their applications to UBP-XNA manipulation. We also discuss the future directions of this field which we consider will involve two-pronged approaches of enzyme biodiscovery for natural UBP-XNA compatible enzymes, coupled with improvement by structure-guided engineering., (© 2025 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Multiple receptor analysis-based DNA molecular computation has been developed to mitigate the off-target effect caused by nonspecific expression of cell membrane receptors. However, it is quite difficult to involve nanobodies into molecular computation with programmed recognition order because of the "always-on" response mode and the inconvenient molecular programming. Here we propose a spatial segregation-based molecular computing strategy with a shielded internal computing layer termed DNA nano-phage (DNP) to program nanobody into DNA molecular computation and build a series of kinetic models to elucidate the mechanism of microenvironment-confinement. We explain the contradiction between fast molecular diffusion and effective DNA computation using a "diffusion trap" theory and comprehensively overcome the kinetic bottleneck of DNP by determining the rate-limiting step. We predict and verify that identifying trace amount of target cells in complex cell mixtures is an intrinsic merit of microenvironment-confined DNA computation. Finally, we show that DNP can efficiently work in complex human blood samples by shielding the interference of erythrocytes and enhance phagocytosis of macrophages toward target cells by blocking CD47-SIRPα pathway., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The DNA-sensing protein cGAS plays a pivotal role in the innate immune response and pathogenesis of various diseases. DNA triggers liquid-liquid phase separation (LLPS) and enhances the enzymatic activity of cGAS. However, the regulatory mechanisms of the disordered N terminus remain unclear. Here, we showed that cGAS Nterm , the N-terminal intrinsic disordered region (IDR) of cGAS, modulates the material properties, specifically the flowability, of the condensed phase of cGAS and is required for full enzymatic activity. Full-length cGAS and cGAS Nterm form liquid droplets in the presence of DNA, while the cGAS catalytic domain forms gel-like solid aggregates with compromised enzymatic activity. Multiple key amino acids responsible for the cGAS Nterm -DNA interaction were identified by NMR spectroscopy as well as other biophysical methods and proven to be critical for the functional LLPS of cGAS both in vitro and in vivo. Interestingly, cGAS Nterm acts in trans to transform the solid aggregates of the cGAS catalytic domain into liquid droplets, subsequently restoring its enzymatic activity. Together, our findings highlight the importance of the IDR of cGAS in LLPS upon DNA stimulation and, more importantly, in modulating the fluidity and permeability of the droplets formed by full-length cGAS, which is crucial for its intact enzymatic activity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cyclic GMP-AMP synthase (cGAS) is a DNA sensing cellular receptor that induces IFN-I transcription in response to pathogen and host derived cytosolic DNA and can limit the replication of some RNA viruses. Some viruses have nonetheless evolved mechanisms to antagonize cGAS sensing. In this study, we evaluated the interaction between Bluetongue virus (BTV), the prototypical dsRNA virus of the Orbivirus genus and the Sedoreoviridae family, and cGAS. We found mitochondrial damage and DNA accumulation in the cytoplasm of infected cells. In addition, we show that BTV infection blocks DNA-induced IFN-I transcription and that virus infection prevents DNA sensing by inducing cGAS and STING degradation. We identify BTV-NS3 as the viral protein responsible for cGAS degradation, showing that NS3 physically interacts with cGAS and induces its degradation through an autophagy-dependent mechanism. Taken together, these findings identify for the first time a mechanism by which a dsRNA virus interferes with a DNA sensing pathway to evade the innate immune response., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Conflict of interest: The authors have no relevant financial or non-financial interest to disclose., (© 2025. The Author(s).)

Stable inheritance of DNA N 6 -methyladenine (6mA) is crucial for its biological functions in eukaryotes. Here, we identify two distinct methyltransferase (MTase) complexes, both sharing the catalytic subunit AMT1, but featuring AMT6 and AMT7 as their unique components, respectively. While the two complexes are jointly responsible for 6mA maintenance methylation, they exhibit distinct enzymology, DNA/chromatin affinity, genomic distribution, and knockout phenotypes. AMT7 complex, featuring high MTase activity and processivity, is connected to transcription-associated epigenetic marks, including H2A.Z and H3K4me3, and is required for the bulk of maintenance methylation. In contrast, AMT6 complex, with reduced activity and processivity, is recruited by PCNA to initiate maintenance methylation immediately after DNA replication. These two complexes coordinate in maintenance methylation. By integrating signals from both replication and transcription, this mechanism ensures the faithful and efficient transmission of 6mA as an epigenetic mark in eukaryotes., Competing Interests: Competing interests statement:The authors declare no competing interest.

We show that a combination of DNA and ionic liquid significantly increases the stability and activity of HRP and achieves a 4.8-fold higher peroxidase activity than PBS buffer. Also, HRP retains 84% of its activity in IL+DNA compared to 24% in PBS against trypsin digestion. Molecular modeling and spectroscopic studies reveal a protective microenvironment.

Two novel mononuclear palladium(II) complexes, [PdL1Cl]Cl (1) and [PdL2Cl]Cl (2) with SNS-donor ligands [where L1 = N -(4-(benzo[ d ]oxazol-2-yl)phenyl)-2-(bis(2-ethylthioethyl)amino)acetamide, L2 = N -(4-(benzo[ d ]thiazol-2-yl)phenyl)-2-(bis(2-ethylthioethyl)amino)acetamide], were synthesized and characterized. In vitro antiproliferative activity tests showed that the two palladium(II) complexes displayed excellent antiproliferative activity against all tested cancer cell lines, especially human colon cancer HCT-116, human liver cancer HepG-2, and human breast cancer MDA-MB-231 cells. Spectacularly, complexes 1 and 2 exhibited approximately 8.49- and 6.88-fold higher antiproliferative activity, as compared with cisplatin, against HCT-116, respectively, but were less toxic to human normal colon fibroblast CCD-18Co cell lines with selectivity index (SI = IC 50 (CCD-18Co)/IC 50 (HCT-116)) values of 22.43 and 21.48 for 1 and 2, respectively, compared to that of cisplatin (SI, 1.74). These results suggested that the two palladium complexes have the potential to act as candidates for the treatment of colorectal cancer. The interaction of the complexes with CT-DNA and pUC19 plasmid DNA illustrated that both 1 and 2 could strongly bind to the DNA helix via an intercalative mode and covalent interaction and perturb the tertiary structure of DNA, where the DNA binding affinity of 1 was slightly higher than that of 2. Additionally, investigations of the reaction of the two complexes with 5'-GMP and glutathione (GSH) showed that both 1 and 2 could readily react with 5'-GMP and GSH to form Pd-GMP adducts and Pd-GS adducts, respectively, and when 5'-GMP and GSH coexisted, the coordination binding of the complexes with GSH did not prevent the formation of the Pd-GMP adducts. Moreover, Hoechst 33342 staining and flow cytometry analysis demonstrated that the two palladium(II) complexes arrested HCT-116 cells mainly at the G2/M phase, induced mitochondrial-membrane depolarization, increased ROS generation, and triggered obvious cell apoptosis.

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long-standing challenge in drug development. MYC is a typical 'undruggable' target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E-Box), and potently inhibit MYC-driven transcription. We crafted the miniproteins via structure-based design and a combination of solid phase peptide synthesis and site-specific crosslinking. Our lead variant, DuoMYC, binds to E-Box DNA with high affinity (K D ~0.1 μM) and is able to enter cells and inhibit MYC-driven transcription with submicromolar potency (IC 50 =464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The century-long Michaelis-Menten rate law and its modifications in the modeling of biochemical rate processes stand on the assumption that the concentration of the complex of interacting molecules, at each moment, rapidly approaches an equilibrium (quasi-steady state) compared to the pace of molecular concentration changes. Yet, in the case of actively time-varying molecular concentrations with transient or oscillatory dynamics, the deviation of the complex profile from the quasi-steady state becomes relevant. A recent theoretical approach, known as the effective time-delay scheme (ETS), suggests that the delay from the relaxation time of molecular complex formation contributes to the substantial breakdown of the quasi-steady state assumption. Here, we systematically expand this ETS and inquire into the comprehensive roles of relaxation dynamics in complex formation. Through the modeling of rhythmic protein-protein and protein-DNA interactions and the mammalian circadian clock, our analysis reveals the effect of the relaxation dynamics beyond the time delay, which extends to the dampening of changes in the complex concentration with a reduction in the oscillation amplitude compared to the quasi-steady state. Interestingly, the combined effect of the time delay and amplitude reduction shapes both qualitative and quantitative oscillatory patterns such as the emergence and variability of the mammalian circadian rhythms. These findings highlight the downside of the routine assumption of quasi-steady states and enhance the mechanistic understanding of rich time-varying biomolecular processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tanshinones, biologically active diterpene compounds derived from Salvia miltiorrhiza , interact with specific proteins and DNA sequences, influencing signaling pathways in animals and humans. This study highlights tanshinone-protein interactions observed at concentrations achievable in vivo, ensuring greater physiological relevance compared to in vitro studies that often employ supraphysiological ligand levels. Experimental data suggest that while tanshinones interact with multiple proteomic targets, only a few enzymes are significantly affected at biologically relevant concentrations. This apparent paradox may be resolved by tanshinones' ability to bind DNA and influence enzymes involved in gene expression or mRNA stability, such as RNA polymerase II and human antigen R protein. These interactions trigger secondary, widespread changes in gene expression, leading to complex proteomic alterations. Although the current understanding of tanshinone-protein interactions remains incomplete, this study provides a foundation for deciphering the molecular mechanisms underlying the therapeutic effects of S. miltiorrhiza diterpenes. Additionally, numerous tanshinone derivatives have been developed to enhance pharmacokinetic properties and biological activity. However, their safety profiles remain poorly characterized, limiting comprehensive insights into their medicinal potential. Further investigation is essential to fully elucidate the therapeutic and toxicological properties of both native and modified tanshinones.

Nucleotide excision repair is a crucial cellular mechanism that ensures genomic stability, thereby preventing mutations that can lead to cancer. The human XPC and its yeast ortholog Rad4 protein complexes are central to this process and were the focus of the study. We used surface plasmon resonance and differential scanning fluorimetry to study the binding characteristics of XPC and Rad4 when bound to the bulky cluster di-FAAF-containing 55-mer duplex DNA. Our findings revealed that XPC binds 10 times more significant affinity to control and di-FAAF-modified DNA than Rad4 with greater protein-DNA interactions. Differential scanning fluorimetry indicates that Rad4 causes comparatively more significant conformational changes upon complexation with the damaged DNA. We conducted DNase I footprinting of the Rad4/DNA complex for the first time by determining the regions protected from DNase I digestion. The DNA at the lesion is entirely resistant to digestion by DNase I in the absence of Rad4 several nucleotides to the 3'-side of the first FAAF lesion. The lack of DNase I cleavage at the lesions did not change upon adding Rad4. However, in the presence of Rad4, a footprint is observed on the 7-nucleotide region (5'-TGGTGAT-3') of the complementary strand to the 3' side of the lesion.

Background: Deoxyribonuclease 2 (DNase II) is pivotal in the clearance of cytoplasmic double stranded DNA (dsDNA). Its deficiency incurs DNA accumulation in cytoplasm, which is a hallmark of multiple neurodegenerative diseases. Our previous study showed that neuronal DNase II deficiency drove tau hyperphosphorylation and neurodegeneration (Li et al., Transl Neurodegener 13:39, 2024). Although it has been verified that DNase II participates in type I interferons (IFN-I) mediated autoinflammation and senescence in peripheral systems, the role of microglial DNase II in neuroinflammation and neurodegenerative diseases such as Alzheimer's disease (AD) is still unknown., Methods: The levels of microglial DNase II in triple transgenic AD mice (3xTg-AD) were measured by immunohistochemistry. The cognitive performance of microglial DNase II deficient WT and AD mice was determined using the Morris water maze test, Y-maze test, novel object recognition test and open filed test. To investigate the impact of microglial DNase II deficiency on microglial morphology, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway and IFN-I pathway, neuroinflammation, synapses loss, amyloid pathology and tauopathy, the levels of cGAS-STING and IFN-I pathway related protein, gliosis and proinflammatory cytokines, synaptic protein, complement protein, Aβ levels, phosphorylated tau in the brains of the microglial DNase II deficient WT and AD mice were evaluated by immunolabeling, immunoblotting, q-PCR or ELISA., Results: We found that the levels of DNase II were significantly decreased in the microglia of 3xTg-AD mice. Microglial DNase II deficiency altered microglial morphology and transcriptional signatures, activated the cGAS-STING and IFN-I pathway, initiated neuroinflammation, led to synapse loss via complement-dependent pathway, increased Aβ levels and tauopathy, and induced cognitive decline., Conclusions: Our study shows the effect of microglial DNase II deficiency and cytoplasmic accumulated dsDNA on neuroinflammation, and reveals the initiatory mechanism of AD pathology, suggesting that DNase II is a potential target for neurodegenerative diseases., Competing Interests: Declarations. Ethics approval and consent to participate: All animal experiments were performed in accordance with the China Public Health Service Guide for the Care and Use of Laboratory Animals. Experiments involving mice and protocols were approved by the Institutional Animal Care and Use Committee of Tsinghua University. Authors are responsible for correctness of the statements provided in the manuscript. Consent for publication: All authors have reviewed the final manuscript and consent to publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Biological polyamines, such as spermine, spermidine, and putrescine, are abundant intracellular compounds mostly bound to nucleic acids. Due to their nucleophilic nature, polyamines easily react with apurinic/apyrimidinic (AP) sites, DNA lesions that are constantly formed in DNA by spontaneous base loss and as intermediates of base excision repair. A covalent intermediate is formed, promoting DNA strand cleavage at the AP site, and is later hydrolyzed regenerating the polyamine. Here we have investigated formation of AP site adducts with spermine and spermidine using sodium borohydride trapping technique and shown that they could persist in DNA for long enough to possibly interfere with cell's replication and transcription machinery. We demonstrate that both adducts placed internally into DNA are strongly blocking for DNA polymerases (Klenow fragment, phage RB69 polymerase, human polymerases β and κ) and direct dAMP incorporation in the rare bypass events. The internal AP site adducts with polyamines can be repaired, albeit rather slowly, by Escherichia coli endonuclease IV and yeast Apn1 but not by human AP endonuclease APE1 or E. coli exonuclease III, whereas the 3'-terminal adducts are substrates for the phosphodiesterase activities of all these AP endonucleases.

DNA interstrand cross-links (ICLs) are the sources of the cytotoxicity of many anticancer agents. Selenium compounds showed great potential as anticancer drugs. In this work, we synthesized a binaphthalene analog 1 containing phenyl selenide (-SePh) as the leaving group and investigated its photochemical reactivity toward DNA as well as its cytotoxicity and selectivity. DNA ICLs were not observed with binaphthalene phenyl selenide 1 without UV irradiation, while ∼15% DNA ICL products were detected with UV irradiation, indicating a photoresponsive property of 1 . The trapping reactions with TEMPO and MeONH 2 , respectively, suggested that free radicals and carbocations are involved in the DNA cross-linking process induced by the photoirradiation of 1 . The photochemical reactivity of 1 toward DNA was sequence-dependent. DNA interstrand cross-linking occurred mainly at dG/dC base pairs, while monoalkylations occurred at dGs and dAs. Additionally, we have demonstrated that 1 alone without UV irradiation did not inhibit cancer cell growth even with a concentration of 100 μM, while the cytotoxicity of 1 toward cancer cells was significantly enhanced upon 350 nm irradiation with an IC 50 of 1.7 μM. No cytotoxicity was observed toward normal epithelial MCF 10A cells, regardless of UV exposure, in the presence or absence of 1 . The alkaline comet assay suggested that the photoinduced cytotoxicity of 1 is correlated to cellular DNA damage. Normal cells showed higher levels of GSH than cancer cells and exhibited efficient DNA repair mechanisms, which can both prevent and repair potential DNA damage induced by 1 , contributing to the selective cytotoxicity of the prodrug toward triple-negative breast cancer cells.

Monitoring enzyme activity is crucial in both scientific research and clinical applications. However, abnormalities in a single enzyme's activity can indicate multiple diseases, limiting the specificity of single enzyme activity monitoring in clinical diagnosis. We developed a dynamic DNA walker that can be sequentially activated by two enzymes, enabling the monitoring and imaging of both enzyme activities within cells. Initially, the DNA walker contains a site for apurinic/apyrimidinic endonuclease 1 (APE1). Upon APE1 activation, the DNA walker forms specific structures recognized and cleaved by Flap endonuclease 1 (FEN1). The temporal disparity between the activities of APE1 and FEN1 allows for the sequential monitoring and imaging of both enzymes, reducing the likelihood of false-positive results. To enhance local concentration and decrease reaction time, the DNA walk sequence was attached to the surface of gold nanoparticles (AuNPs). The fruition of this endeavor will facilitate the investigation and advancement of multiple enzyme activity monitoring and imaging methods and technologies, while simultaneously broadening the domains of application for DNA nanotechnology.

Background/objectives: Predicting the effects of protein and DNA mutations on the binding free energy of protein-DNA complexes is crucial for understanding how DNA variants impact wild-type cellular function. As many cellular interactions involve protein-DNA binding, accurately predicting changes in binding free energy (ΔΔG) is valuable for distinguishing pathogenic mutations from benign ones., Methods: This study describes the development and optimization of the SAMPDI-3Dv2 machine learning method, which is trained on an expanded database of experimentally measured ΔΔGs. This enhanced model incorporates new features, including the 3D structure of the mutant protein, features of the mutant structure, and a position-specific scoring matrix (PSSM). Benchmarking was conducted using 5-fold cross-validation., Results: The updated SAMPDI-3D model (SAMPDI-3Dv2) achieved Pearson correlation coefficients (PCCs) of 0.68 for protein and 0.80 for DNA mutations. These results represent significant improvements over existing tools. Additionally, the method's rapid execution time enables genome-scale predictions., Conclusions: The improved SAMPDI-3Dv2 shows enhanced predictive performance for analyzing mutations in protein-DNA complexes. By leveraging structural information and an expanded training dataset, SAMPDI-3Dv2 provides researchers with a more accurate and efficient tool for mutation analysis, contributing to identifying pathogenic variants and improving our understanding of cellular function.

Type IA topoisomerases (TopoIAs) are present in all living organisms. They resolve DNA/RNA catenanes, knots and supercoils by breaking and rejoining single-stranded DNA/RNA segments and allowing the passage of another nucleic acid segment through the break. Topoisomerase III-β (TOP3B), the only RNA topoisomerase in metazoans, promotes R-loop disassembly and translation of mRNAs. Defects in TOP3B lead to severe neurological diseases. We present a series of cryo-EM structures of human TOP3B with its cofactor TDRD3 during cleavage and rejoining of DNA or RNA, thus elucidating the roles of divalent metal ions and key enzyme residues in each step of the catalytic cycle. We also obtained the structure of an open-gate configuration that addresses the long-standing question of the strand-passage mechanism. Our studies reveal how TOP3B catalyzes both DNA and RNA relaxation, while TOP3A acts only on DNA., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

The MCM motor of the eukaryotic replicative helicase is loaded as a double hexamer onto DNA by the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ATP binding supports formation of the ORC-Cdc6-Cdt1-MCM (OCCM) helicase-recruitment complex where ORC-Cdc6 and one MCM hexamer form two juxtaposed rings around duplex DNA. ATP hydrolysis by MCM completes MCM loading but the mechanism is unknown. Here, we used cryo-EM to characterise helicase loading with ATPase-dead Arginine Finger variants of the six MCM subunits. We report the structure of two MCM complexes with different DNA grips, stalled as they mature to loaded MCM. The Mcm2 Arginine Finger-variant stabilises DNA binding by Mcm2 away from ORC/Cdc6. The Arginine Finger-variant of the neighbouring Mcm5 subunit stabilises DNA engagement by Mcm5 downstream of the Mcm2 binding site. Cdc6 and Orc1 progressively disengage from ORC as MCM translocates along DNA. We observe that duplex DNA translocation by MCM involves a set of leading-strand contacts by the pre-sensor 1 ATPase hairpins and lagging-strand contacts by the helix-2-insert hairpins. Mutating any of the MCM residues involved impairs high-salt resistant DNA binding in vitro and double-hexamer formation assessed by electron microscopy. Thus, ATPase-powered duplex DNA translocation away from ORC underlies MCM loading., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The DNA adducts formed by the alkenylbenzene natural products, safrole (SF) and methyleugenol (MEG) are primarily attributed to their reported carcinogenic properties. Herein, we report a concise strategy to access N 6 -Ac-SF/MEG-dA phosphoramidites, which were selectively incorporated into DNA oligonucleotides by solid-phase DNA synthesis. The replication studies using human polymerases hpolκ and hpolη showed that both polymerases replicate these adducts error-free, which indicates that these polymerases do not contribute to the adduct-induced mutagenicity.