DNA Binding Properties Research Articles

Crystal Structure and DNA Binding Properties of A Sulfide Bridged Dimeric Schiff Base Compound

In this study, a new 4,4'-Diaminodiphenyl sulfide-based Schiff base compound [6,6'-((1E,1'E)-((thiobis (4,1-phenylene)) bis(azaneylylidene)) bis(methaneylylidene)) bis(3-(diethylamino) phenol) (A)], which is known to have very good biological activity and form the basis of anticancer drugs, was successfully synthesized. The spectral properties and DNA binding properties of the Schiff base compound obtained within the scope of the study were investigated. The structure of the synthesized compound was characterized by UV–Vis, FT-IR, 1H NMR, 13C NMR and Photoluminescence spectroscopy. However, the molecular structure of the synthesized compound was determined by single-crystal X-ray diffraction study.

EIN3/EIL (Ethylene Insensitive3 / Ethylene Insensitive3 Like) Protein Family in Phaseolus vulgaris: Identification, Evolution and Expression Analysis within the Genome

Ethylene insensitive-3 (EIN3) / Ethylene insensitive-3-like (EIL) protein family is a small family of transcription factors specific to plants that play role in plant growth and development under various environmental conditions. In this study, various bioinformatics approaches were used to make an in-depth identification of the EIN3/EIL family at both the gene and protein levels. So, 11 Pvul-EIL genes were identified and their approximate locations were determined. Various biochemical and physicochemical properties of EIL proteins in Phaseolus vulgaris have been described. It was determined that Pvul-EIL proteins had a length of 447-651 amino acids and a molecular weight of 51.08-70.68 kDa. All duplications occurring in the Pvul-EIL genome were segmental type. It was observed that conserved motif, gene structure and phylogeny analyses all yielded similar results. For instance, it has been understood that genes with same motif type and number have similar gene structures and were located under the same branch in the phylogenetic tree. Pvul-EIL protein homology modeling showed that DNA binding properties and protein structure were similar to Arabidopsis EIN3. According to cis-element analysis, Pvul-EIL genes are engaged in a wide range of functions, including tissue-specific, stress, and hormone-sensitive expression. Additionally, RNAseq data was used to perform a comparative expression analysis of EIL genes. Various Pvul-EIL gene expression levels were detected under salt and drought stress. This is the first study to check the gene expression levels in P. vulgaris using in-silico detection and characterization of EIL genes. Therefore, obtained results can form the basis for future studies.

Spectroscopy, docking and molecular dynamics studies on the interaction between cis and trans palladium-alanine complexes with calf-thymus DNA and antitumor activities

The present article investigates whether the cis and trans isomers of the same compound affect their antitumor and DNA-binding properties. Thus, we have synthesized cis-[Pd(L-Ala)2] (I) and trans-[Pd(L-Ala)2] (II) (Ala = L-alanine) complexes by modifications to improve the reported procedure. Cytotoxicity tests of these two isomers along with carboplatin (as a known drug) against MCF-7 and HCT-116 (as cancerous) and MCF10A and CCD-841 (as normal cell lines) were performed under the same experimental conditions. The results follow the sequence I > II > carboplatin for both cell lines. DNA-binding studies were carried out in a medium similar to that of physiological conditions (pH = 7 and 0.02 M NaCl). Results of UV–vis absorption provides apparent binding constant, K app, and midpoint transition of native CT-DNA to refolded structure, L1/2, suggesting interaction at quite low concentrations of these metal complexes. Fluorescence emission intensity of the CT-DNA-EB system demonstrates static quenching predominates in the binding process. Moreover, negative values of ΔH 0 and ΔS 0 suggested that the hydrogen bonds and van der Waals forces are the interaction forces between the complexes with the CT-DNA groove. Other studies include gel electrophoresis, viscosity experiments, partition coefficient, computational energy minimization, molecular docking and molecular dynamics simulations. All these studies indicate that the cis isomer has better interaction towards CT-DNA and are in accord with fluorescence, UV–vis and cytotoxicity studies. The present work gives valuable indications for further design of cis and trans metal-based isomers as potential anticancer agents.

A comparative study on DNA and protein binding properties of thymol and thymoquinone

Two phytochemicals, thymol and thymoquinone obtained from thymes (Thymus vulgaris L., Lamiaceae etc.) and Nagila Sativa seed, respectively. Both the phytochemicals show several biochemical activities like anticancer, antimicrobial etc. In this paper, we studied the affinities of thymol and thymoquinone towards calf thymus DNA (CT-DNA) and protein (bovine serum albumin). Spectroscopic and molecular modelling studies revealed that both compounds have a high affinity toward both the receptors; DNA and protein. Both phytochemicals binds to the minor grooves of DNA and suitable pockets of protein. Several free energy function and hydrogen bonding play significant role during the binding phenomenon. Communicated by Ramaswamy H. Sarma

A fluorene based imine compound: Structural characterization, DNA binding properties and fluorescence sensor properties towards metal ions

In the course of this work, a new fluorene-based Schiff base compound (SB) from the condensation reaction 2-aminofluorene and 4-(diethylamino)salicylaldehyde was synthesized and its structure was characterized by FTIR, 1H/13C NMR spectroscopies and elemental analysis. Crystal structure of the synthesized compound (SB) was determined single crystal X-ray diffraction study. Crystal structure of the compound was solved in monoclinic unit cell and P21 space group. The molecule shows phenol-imine intramolecular hydrogen bond and molecules are connected by CH····π interactions. The Schiff base compound was then screened for its DNA binding properties by using UV–vis absorption, fluorescence and viscosity studies. All DNA binding data showed that the compound interacts with FSdsDNA via intercalation. The intrinsic DNA binding constant of the compound from UV–vis spectral data was 3.00×104 M−1 which is significantly lower than ethidium bromide (an intercalating agent). The compound was further investigated for colorimetric sensing of metal ions. Colorimetric sensor properties of the compound towards Fe(III), Pb(II), Zn(II), Cu(II), Al(III), Hg(II), Co(II), Mn(II) and Ni(II) ions were investigated in solution. The compound showed selective sensing properties for Cu(II) under 365 nm light. Jobs variation study indicated that the Cu(II) to receptor ratio was 2:1.

Nucleus size and its effect on nucleosome stability in living cells.

Spatial conformation of chromatin in eukaryotic cells is mainly determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints imposed on chromosomal DNA by a tight nuclear space. Yet, the exact effect of the nucleus size on DNA-protein interactions and chromatin structure currently remains obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. In our study, we analyse the relative contribution of several key components of living cells to the regulation of the nucleus size through direct application of pressure on the nuclear envelope. It is shown that the nucleus size in higher eukaryotes is mainly defined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, calculations demonstrate that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.

Controlled modulation of the dynamics of the Deinococcus grandis Dps N-terminal tails by divalent metals.

DNA-binding proteins from starved cells (Dps) are small multifunctional nanocages expressed by prokaryotes in acute oxidative stress conditions or during the starvation-induced stationary phase, as a bacterial defense mechanism. Dps proteins protect bacterial DNA from damage by either direct binding or by removing precursors of reactive oxygen species from solution. The DNA-binding properties of most Dps proteins studied so far are related to their unordered, flexible, N- and C-terminal extensions. In a previous work, we revealed that the N-terminal tails of Deinoccocus grandis Dps shift from an extended to a compact conformation depending on the ionic strength of the buffer and detected a novel high-spin ferrous iron center in the proximal ends of those tails. In this work, we further explore the conformational dynamics of the protein by probing the effect of divalent metals binding to the tail by comparing the metal-binding properties of the wild-type protein with a binding site-impaired D34A variant using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism, and small-angle X-ray scattering. The N-terminal ferrous species was also characterized by Mössbauer spectroscopy. The results herein presented reveal that the conformation of the N-terminal tails is altered upon metal binding in a gradual, reversible, and specific manner. These observations may point towards the existence of a regulatory process for the DNA-binding properties of Dps proteins through metal binding to their N- and/or C-terminal extensions.

Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding

Cys2His2 zinc finger proteins are important for living organisms, as they—among other functions—specifically recognise DNA when Zn(II) is coordinated to the proteins, stabilising their ββα secondary structure. Therefore, competition with other metal ions may alter their original function. Toxic metal ions such as Cd(II) or Hg(II) might be especially dangerous because of their similar chemical properties to Zn(II). Most competition studies carried out so far have involved small zinc finger peptides. Therefore, we have investigated the interactions of toxic metal ions with a zinc finger proteins consisting of three finger units and the consequences on the DNA binding properties of the protein. Binding of one Cd(II) per finger subunit of the protein was shown by circular dichroism spectroscopy, fluorimetry and electrospray ionisation mass spectrometry. Cd(II) stabilised a similar secondary structure to that of the Zn(II)-bound protein but with a slightly lower affinity. In contrast, Hg(II) could displace Zn(II) quantitatively (logβ′ ≥ 16.7), demolishing the secondary structure, and further Hg(II) binding was also observed. Based on electrophoretic gel mobility shift assays, the Cd(II)-bound zinc finger protein could recognise the specific DNA target sequence similarly to the Zn(II)-loaded form but with a ~0.6 log units lower stability constant, while Hg(II) could destroy DNA binding completely.

Synergy of ruthenium metallo-intercalator, [Ru(dppz)2(PIP)]2+, with PARP inhibitor Olaparib in non-small cell lung cancer cells

Poly(ADP-ribose) polymerase (PARP) are critical DNA repair enzymes that are activated as part of the DNA damage response (DDR). Although inhibitors of PARP (PARPi) have emerged as small molecule drugs and have shown promising therapeutic effects, PARPi used as single agents are clinically limited to patients with mutations in germline breast cancer susceptibility gene (BRCA). Thus, novel PARPi combination strategies may expand their usage and combat drug resistance. In recent years, ruthenium polypyridyl complexes (RPCs) have emerged as promising anti-cancer candidates due to their attractive DNA binding properties and distinct mechanisms of action. Previously, we reported the rational combination of the RPC DNA replication inhibitor [Ru(dppz)2(PIP)]2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine, PIP = 2-(phenyl)-imidazo[4,5-f][1,10]phenanthroline), “Ru-PIP”, with the PARPi Olaparib in breast cancer cells. Here, we expand upon this work and examine the combination of Ru-PIP with Olaparib for synergy in lung cancer cells, including in 3D lung cancer spheroids, to further elucidate mechanisms of synergy and additionally assess toxicity in a zebrafish embryo model. Compared to single agents alone, Ru-PIP and Olaparib synergy was observed in both A549 and H1975 lung cancer cell lines with mild impact on normal lung fibroblast MRC5 cells. Employing the A549 cell line, synergy was confirmed by loss in clonogenic potential and reduced migration properties. Mechanistic studies indicated that synergy is accompanied by increased double-strand break (DSB) DNA damage and reactive oxygen species (ROS) levels which subsequently lead to cell death via apoptosis. Moreover, the identified combination was successfully able to inhibit the growth of A549 lung cancer spheroids and acute zebrafish embryos toxicity studies revealed that this combination showed reduced toxicity compared to single-agent Ru-PIP.

Synthesis, crystal structures and DNA-binding properties of Zn(II), Cd(II), Mn(II), Cu(II) complexes based on a thiazole derivative and carboxylic acids

By single-crystal X-ray diffraction (XRD) analysis, five complexes, catena-[(µ-2,2-bis(4-carboxyphenyl)hexafluoropropane)-(2-(pyidin-2-yl)-1,3-benzothiazole)-aqua-zinc(II)] (1), catena-[(µ-2,2-bis(4-carboxyphenyl)hexafluoropropane)-(2-(pyidin-2-yl)1,3-benzothiazole)-aqua-cadmium(II)] (2), catena-[hepta(µ-2,2-bis(4carboxyphenyl)hexafluoropropane)-diaqua-bis(2-(pyidin-2-yl)-1,3-benzothiazole)-tri-manganes(II)] (3), catena-[(µ-benzene-1,2,4,5-tetracarboxylato)-(2-(pyidin-2-yl)-1,3benzothiazole)copper(II)] (4) and catena-[(µ-3-nitrobenzene-1,2-dicarboxylato)-(2-(pyidin-2-yl)1,3-benzothiazole)-aqua-zinc(II)] (5), were characterized in this research. In these five complexes, H2hfipbb = 2,2-bis(4-carboxyphenyl)hexafluoropropane, H4btec = 1,2,4,5-benzenetetracarboxylic acid, H2NPTA = 3-nitrophthalic acid and bpt = 2-(pyridin-2-yl)benzo[d]thiazole). Complexes 1 and 2 occur in the monoclinic system with space group C2/c, 4 and 5 occur in the monoclinic system with space group P21/c, and 3 occurs in the triclinic system with space group P-1. The interaction between CT-DNA (calf thymus DNA) and those complexes was examined using UV spectroscopic analysis, fluorescent spectroscopic analysis, circular dichroism spectroscopy (CD) and viscosity analysis. This research reveals that those complexes have strong ability to interact with DNA, and they all bind to DNA in an intercalation mode, thus affecting the structure of DNA. This binding mode can arise from the interaction between the benzothiazole metal complexes and DNA. Hence, we hope to provide some scientific research bases for studying benzothiazole complexes and designing new thiazole anticancer drugs.

Ruthenium(II) Polypyridyl Complexes as FRET Donors: Structure- and Sequence-Selective DNA-Binding and Anticancer Properties.

Ruthenium(II) polypyridyl complexes (RPCs) that emit from metal-to-ligand charge transfer (MLCT) states have been developed as DNA probes and are being examined as potential anticancer agents. Here, we report that MLCT-emissive RPCs that bind DNA undergo Förster resonance energy transfer (FRET) with Cy5.5-labeled DNA, forming mega-Stokes shift FRET pairs. Based on this discovery, we developed a simple and rapid FRET binding assay to examine DNA-binding interactions of RPCs with diverse photophysical properties, including non-"light switch" complexes [Ru(dppz)2(5,5'dmb)]2+ and [Ru(PIP)2(5,5'dmb)]2+ (dppz = dipyridophenazine, 5,5'dmb = 5,5'-dimethyl-2,2'-bipyridine, PIP = 2-phenyl-imidazo[4,5-f][1,10]phenanthroline). Binding affinities toward duplex, G-quadruplex, three-way junction, and mismatch DNA were determined, and derived FRET donor-acceptor proximities provide information on potential binding sites. Molecules characterized by this method demonstrate encouraging anticancer properties, including synergy with the PARP inhibitor Olaparib, and mechanistic studies indicate that [Ru(PIP)2(5,5'dmb)]2+ acts to block DNA replication fork progression.

Switching between DNA binding modes with a photo- and redox-active DNA-targeting ligand.

A disulfide-functionalized bis-benzo[b]quinolizinium is presented that is transformed quantitatively into its cyclomers in a fast intramolecular [4 + 4] photocycloaddition. Both the bis-quinolizinium and the photocyclomers react with glutathione (GSH) or dithiothreitol (DTT) to give 9-(sulfanylmethyl)benzo[b]quinolizinium as the only product. As all components of this reaction sequence have different DNA-binding properties, it enables the external control and switching of DNA association. Hence, the bis-benzo[b]quinolizinium binds strongly to DNA and is deactivated upon photocycloaddition to the non-binding cyclomers. In turn, the subsequent cleavage of the cyclomers with DTT regains a DNA-intercalating benzoquinolizinium ligand. Notably, this sequence of controlled deactivation and recovery of DNA-binding properties can be performed directly in the presence of DNA.

Assessing TFAM Binding to Human Mitochondrial DNA.

Mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA)-binding protein that plays a crucial dual role in the initiation of mitochondrial transcription initiation and mtDNA maintenance. Because TFAM directly interacts with mtDNA, assessing its DNA-binding property can provide useful information. This chapter describes two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay with recombinant TFAM proteins, which both require simple agarose gel electrophoresis. These are used to investigate the effects of mutations, truncation, and posttranslational modifications on this key mtDNA regulatory protein.

Switching between DNA binding modes with a photo- and redox-active DNA-targeting ligand, part II: the influence of the substitution pattern.

A disulfide-functionalized photoactive DNA ligand is presented that enables the control of its DNA-binding properties by a combination of a photocycloaddition reaction and the redox reactivity of the sulfide/disulfide functionalities. In particular, the initially applied ligand binds to DNA by a combination of intercalation and groove-binding of separate benzo[b]quinolizinium units. The association to DNA is interrupted by an intramolecular [4 + 4] photocycloaddition to the non-binding head-to-head cyclomers. In turn, the subsequent cleavage of these cyclomers with dithiothreitol (DTT) regains temporarily a DNA-intercalating benzoquinolizinium ligand that is eventually converted into a non-binding benzothiophene. As a special feature, this sequence of controlled deactivation, recovery and internal shut-off of DNA-binding properties can be performed directly in the presence of DNA.

G-quadruplex DNA binding properties of novel nickel Schiff base complexes with four pendant groups.

Three new nickel Schiff base complexes were prepared using a two-step procedure that involves initial selective dialkylation of 2,4,6-trihydroxybenzaldehyde, followed by reaction with 1,2-phenylenediamine and nickel(II) acetate. Each of the complexes possessed the same Schiff base core but differed in the identity of the four pendant groups. All complexes were characterised by microanalysis, NMR spectroscopy and ESI mass spectrometry. In addition, two of the complexes were also characterised in the solid state using X-ray crystallography, which confirmed the presence of a square planar geometry around the metal ion. The DNA binding properties of the three nickel complexes with double stranded DNA and a range of G-quadruplex DNA structures were explored using ESI mass spectrometry, CD spectroscopy, UV melting curves, Fluorescence Resonance Energy Transfer (FRET) assays, Fluorescent Indicator Displacement (FID) assays and molecular docking studies. These techniques confirmed the ability of the three nickel complexes to bind to most of the DNA molecules examined, as well as stabilise the latter in several instances. In addition, the results of these investigations provided evidence that pendant groups with morpholine rings generally reduced DNA binding behaviour, whilst pendants featuring piperidine ring systems attached to the Schiff base core by three and not two methylene linkers often showed the greatest extent of binding or DNA stabilisation.

Structural characterization and DNA binding properties of a phenanthrene based Schiff base compound

Fenantren-9-karbaldehit ve 4-aminofenolün kondenzasyon reaksiyonu ile fenantren bazlı yeni bir Schiff baz bileşiği (PBS) sentezlenmiştir. Bileşiğin yapısı FTIR, 1H(13C) NMR ve elementel analizi ile karakterize edildi. Bileşiğin kristal yapısı, tek kristal X-ışını kırınım deneyi ile belirlendi. X-ışını kristalografik verileri, fenantren ve fenol halkalarının birbirine göre yaklaşık olarak dik olduğunu ortaya çıkardı. Yapıda moleküller, moleküller arası fenol-imin hidrojen bağları [O1-H·····N1] ile bağlanır ve supramoleküler hidrojen bağ zincirleri oluşturur. Ayrıca fenantren halka sistemi, komşu moleküllerin aynı bölümleri ile π-π istifleme etkil (baş-kuruk) etkileşimlerinde yer alır. Bileşiğin çift sarmallı balık sperm DNA'sına (dsFS-DNA) bağlanma özellikleri spektrofotometrik, florimetrik ve viskosimetrik yöntemlerle araştırılmıştır. Spektral veriler, bileşiğin, önemli bağlanma sabiti (Kb: 4.6 x 104 M-1) ile oluk bağlanma modunda DNA ile etkileşime girdiğini göstermiştir. Bileşik PBS'nin varlığında, dsFS-DNA'nın viskozitesi önemli bir değişiklik gözlenmemesi, bileşiğin dsFS-DNA ile interkalatif olmayan bağlanma modunda etkileşime girdiğini göstermektedir.

Design, synthesis, spectroscopic characterizations, single crystal X-ray analysis, in vitro xanthine oxidase and acetylcholinesterase inhibitory evaluation as well as in silico evaluation of selenium-based N-heterocyclic carbene compounds

Herein, eight new NHC-based selenourea derivatives were synthesized and characterized by using spectroscopic method (1H, 19F, and 13C NMR, FT-IR), and elemental analysis techniques. These compounds were synthesized by mixing benzimidazolium salts, potassium carbonate, and selenium powder in ethyl alcohol. Additionally, the molecular and crystal structures of the three compounds (1c, 2b, and 2c) were determined using the single-crystal x-ray diffraction (XRD) method. Diffraction analysis demonstrated the partial carbon-selenium double-bond character of these compounds. All compounds were determined to be highly potent inhibitors for AChE and XO enzymes. The IC50 values for the compounds were found in the range of 0.361–0.754 μM for XO and from 0.995 to 1.746 μM for AChE. The DNA binding properties of the compounds were investigated. These compounds did not have a remarkable DNA binding property. Also, DPPH radical scavenging activities of the compounds were also investigated. Compounds (1c), (2a), (3a), and (3b) exhibited more pronounced DPPH radical scavenging activity when compared to other compounds. Docking studies were applied by using AutoDock 4 to determine interaction mechanism of the selected compounds (1a), (1b), and (3b). The compound (1b) has good binding affinity (−9.78 kcal/mol) against AChE, and (−6.86 kcal/mol) for XO target. Drug similarity properties of these compounds compared to positive controls were estimated and evaluated by ADMET analysis. Furthermore, molecular dynamics simulations have been applied to understand the accuracy of docking studies. These findings and the defined compounds could be potential candidates for the discovery and progress of effective medicine(s) for AChE and XO in the future. Communicated by Ramaswamy H. Sarma

One-pot synthesis of cyclic-aminotropiminium carboxylate derivatives with DNA binding and anticancer properties

Tropolone, a nonbenzenoid aromatic molecule, is a constituent of troponoid natural products possessing a wide range of bioactivities, including anticancer. This report describes the one-pot synthesis and mechanistic studies of fifteen fluorescent Caryl-Nalkyl-substituted cyclic-aminotroponiminium carboxylate (cATC) derivatives by unusual cycloaddition and rearrangement reactions. Herein, the biochemical studies of four cATC derivatives reveal a non-intercalative binding affinity with DNA duplex. In vitro/in vivo studies show strong anti-tumor activity in three cATC derivatives. These derivatives enter the cells and localize to the nucleus and cytoplasm, which are easily traceable due to their inherent fluorescence properties. These three cATC derivatives reduce the proliferation and migration of HeLa cells more than the non-cancer cell line. They induce p38-p53-mediated apoptosis and inhibit EMT. In xenograft-based mouse models, these cATC derivatives reduce tumor size. Overall, this study reports the synthesis of DNA binding fluorescent Caryl-Nalkyl-cyclic-aminotroponiminium derivatives which show anti-tumor activity with the minimum side effect.

Hypoxia-Inducible Factor-1: A Novel Therapeutic Target for the Management of Cancer, Drug Resistance, and Cancer-Related Pain.

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates the transcription of many genes that are responsible for the adaptation and survival of tumor cells in hypoxic environments. Over the past few decades, tremendous efforts have been made to comprehensively understand the role of HIF-1 in tumor progression. Based on the pivotal roles of HIF-1 in tumor biology, many HIF-1 inhibitors interrupting expression, stabilization, DNA binding properties, or transcriptional activity have been identified as potential therapeutic agents for various cancers, yet none of these inhibitors have yet been successfully translated into clinically available cancer treatments. In this review, we briefly introduce the regulation of the HIF-1 pathway and summarize its roles in tumor cell proliferation, angiogenesis, and metastasis. In addition, we explore the implications of HIF-1 in the development of drug resistance and cancer-related pain: the most commonly encountered obstacles during conventional anticancer therapies. Finally, the current status of HIF-1 inhibitors in clinical trials and their perspectives are highlighted, along with their modes of action. This review provides new insights into novel anticancer drug development targeting HIF-1. HIF-1 inhibitors may be promising combinational therapeutic interventions to improve the efficacy of current cancer treatments and reduce drug resistance and cancer-related pain.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.