DNA Binding Mode Research Articles

DNA–TCP complex structures reveal a unique recognition mechanism for TCP transcription factor families

Plant-specific TCP transcription factors are key regulators of diverse plant functions. TCP transcription factors have long been annotated as basic helix-loop-helix (bHLH) transcription factors according to remote sequence homology without experimental validation, and their consensus DNA-binding sequences and protein-DNA recognition mechanisms have remained elusive. Here, we report the crystal structures of the class I TCP domain from AtTCP15 and the class II TCP domain from AtTCP10 in complex with different double-stranded DNA (dsDNA). The complex structures reveal that the TCP domain is a distinct DNA-binding motif and the homodimeric TCP domains adopt a unique three-site recognition mode, binding to dsDNA mainly through a central pair of β-strands formed by the dimer interface and two basic flexible loops from each monomer. The consensus DNA-binding sequence for class I TCPs is a perfectly palindromic 11 bp (GTGGGNCCCAC), whereas that for class II TCPs is a near-palindromic 11 bp (GTGGTCCCCAC). The unique DNA binding mode allows the TCP domains to display broad specificity for a range of DNA sequences even shorter than 11 bp, adding further complexity to the regulatory network of plant TCP transcription factors.

Structure of monkeypox virus DNA polymerase holoenzyme

The World Health Organization declared mpox (or monkeypox) a public health emergency of international concern in July 2022, and prophylactic and therapeutic measures are in urgent need. The monkeypox virus (MPXV) has its own DNA polymerase F8, together with the processive cofactors A22 and E4, constituting the polymerase holoenzyme for genome replication. Here, we determined the holoenzyme structure in complex with DNA using cryo-electron microscopy at the global resolution of ~2.8 angstroms. The holoenzyme possesses an architecture that suggests a "forward sliding clamp" processivity mechanism for viral DNA replication. MPXV polymerase has a DNA binding mode similar to that of other B-family DNA polymerases from different species. These findings reveal the mechanism of the MPXV genome replication and may guide the development of anti-poxvirus drugs.

Two Distinct Modes of DNA Binding by an MCM Helicase Enable DNA Translocation.

A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism Saccharolobus solfataricus binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation.

MutS recognition of mismatches within primed DNA replication intermediates

MutS initiates mismatch repair by recognizing mismatches in newly replicated DNA. Specific interactions between MutS and mismatches within double-stranded DNA promote ADP-ATP exchange and a conformational change into a sliding clamp. Here, we demonstrated that MutS from Pseudomonas aeruginosa associates with primed DNA replication intermediates. The predicted structure of this MutS-DNA complex revealed a new DNA binding site, in which Asn 279 and Arg 272 appeared to directly interact with the 3′-OH terminus of primed DNA. Mutation of these residues resulted in a noticeable defect in the interaction of MutS with primed DNA substrates. Remarkably, MutS interaction with a mismatch within primed DNA induced a compaction of the protein structure and impaired the formation of an ATP-bound sliding clamp. Our findings reveal a novel DNA binding mode, conformational change and intramolecular signaling for MutS recognition of mismatches within primed DNA structures.

Molecular basis of the key regulator WRINKLED1 in plant oil biosynthesis.

Vegetable oils are not only major components of human diet but also vital for industrial applications. WRINKLED1 (WRI1) is a pivotal transcription factor governing plant oil biosynthesis, but the underlying DNA-binding mechanism remains incompletely understood. Here, we resolved the structure of Arabidopsis WRI1 (AtWRI1) with its cognate double-stranded DNA (dsDNA), revealing two antiparallel β sheets in the tandem AP2 domains that intercalate into the adjacent major grooves of dsDNA to determine the sequence recognition specificity. We showed that AtWRI1 represented a previously unidentified structural fold and DNA-binding mode. Mutations of the key residues interacting with DNA element affected its binding affinity and oil biosynthesis when these variants were transiently expressed in tobacco leaves. Seed oil content was enhanced in stable transgenic wri1-1 expressing an AtWRI1 variant (W74R). Together, our findings offer a structural basis explaining WRI1 recognition and binding of DNA and suggest an alternative strategy to increase oil yield in crops through WRI1 bioengineering.

Synthesis of Mn-ofloxacin complex, experimental and in silico DNA binding evaluation, biological activity assessment

Metal-fluoroquinolones have more antibacterial and cytotoxic effects compared to free fluoroquinolones. In this work, a bidentated Mn (II) complex with ofloxacin (MOC) was synthesized and its cytotoxicity activity, oxidative stress and DNA binding were studied. Anti- proliferative and cytotoxic tests revealed that MOC exhibits better anti proliferative and cytotoxic activities compared to OFL which was attributed to the more interaction of MOC with DNA. Therefore, the interaction of MOC with DNA was investigated by using voltammetry, UV-Vis, fluorescence, and in silico methods. The results revealed that MOC interacts with DNA via electrostatic and outside hydrogen binding via minor groove. The proposed DNA binding modes may support the greater in-vitro cytotoxicity of MOC compared to OFL alone.

Structures of topoisomerase V in complex with DNA reveal unusual DNA-binding mode and novel relaxation mechanism.

Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.

Elucidation of binding interactions and mechanism of rivastigmine tartrate with dsDNA via multi-spectroscopic, electrochemical, and molecular docking studies

Rivastigmine tartrate (RT) is a noncompetitive carbamate acetylcholinesterase inhibitor that is carbamylated, long-acting, and reversible. It's used to alleviate Alzheimer's disease symptoms that range from mild to moderate. Although various cholinesterase inhibitors have been researched for their DNA binding mode, the RT-dsDNA binding mechanism has not been investigated yet. As a result, both experimentally and theoretically, the binding mechanism and reversible interactions of RT with double-stranded fish sperm deoxyribonucleic acid (dsDNA) have been thoroughly investigated. Our overall purpose is to have a better understanding of the potential of cholinesterase inhibitor medications in order to find novel medications that target dsDNA and develop drugs with fewer adverse effects. We applied a variety of spectroscopic methods under physiological situations, including UV-vis and fluorescence, spectroscopy, thermal denaturation, electrochemical and viscosity measurements, as well as molecular docking investigations. In addition, the interactions between RT and dsDNA were investigated computationally with the assistance of molecular docking and molecular dynamics simulations. Data from both experimental and theoretical results showed that RT interacts with dsDNA through the groove binding mode.

Preliminary in Silico Studies of the Interactions of Certain Genotoxic Azo Dyes with Different Double-Stranded DNA Conformations

Organic azo dyes, which are widely used in industrial, health and cosmetic fields, pose genotoxic risks due to their chemical structures; however, the molecular details of the undesirable effects of these dyes on DNA have been poorly or insufficiently clarified. In this computational molecular docking study, the DNA binding modes and binding affinities of 14 azo dyes, previously determined to show DNA clastogenicity, were characterized using 2 different double-stranded DNA (dsDNA) conformations (an intact dsDNA and dsDNA with an intercalation gap). In this study, it was determined that 10 out of the 14 genotoxic azo dyes were strong dsDNA minor groove binders, while the remaining ones formed tight binding complexes with dsDNA through intercalation or threading intercalation modes. The azo, nitro, hydroxyl, ammonium, sulfonate, naphthalene, methoxyphenyl, bromine, nitrophenyl, imidazole, amino-phenylethanol and chloro-nitrophenyl groups were found to play primary role in the most favorable binding conformations of these dyes on dsDNA with an affinity ranging from −6.35 kcal/mol to −9.42 kcal/mol. It was determined that dsDNA sequences containing GT dinucleotides are frequently preferred in binding by these dyes, and that rings and polar groups are important features for tight binding with dsDNA. It was concluded that these dyes may be banned, or non-genotoxic congeners should be manufactured with appropriate molecular optimization for the genetic health of the human population and for future generations.

Mixed Ligand Mononuclear Copper(II) Complex as a Promising Anticancer Agent: Interaction Studies with DNA/HSA, Molecular Docking, and In Vitro Cytotoxicity Studies.

The isolated copper(II) complex [CuL(o-phen)]·H2O (1) [H2L = o-HO-C6H4C(H)=N-C6H4-SH-o, o-phen = 1,10-phenanthroline] was structurally characterized using single-crystal X-ray crystallography. 1 in CH3CN at liquid nitrogen temperature displayed a characteristic monomeric X-band electron paramagnetic resonance spectrum having a tetragonal character with g∥ = 2.1479 and g⊥ = 2.0691 and A∥ ≈ 18.0 mT and A⊥ ≤ 3.9 mT, respectively. 1 showed a strong binding affinity toward calf thymus DNA as reflected from its intrinsic binding constant (Kb = 7.88 × 105 M–1), and its competitive displacement of ethidium bromide suggested an intercalative DNA-binding mode (Kapp = 1.32 × 106 M–1). This was confirmed from the viscosity study that showed an increase in the viscosity of DNA with an increasing concentration of 1. Complex 1 is highly efficient in promoting oxidative and hydrolytic DNA cleavage (kobs = 1.987 h–1). 1 showed a strong binding affinity with the carrier protein human serum albumin (HSA) (Ka = 5.22 × 105 M–1). A high bimolecular quenching constant kq = 2.29 × 1013 M–1s–1 indicated a static quenching mechanism involved in the fluorescence quenching of HSA by 1. Fluorescence resonance energy transfer theory suggested that the distance (r = 3.52 nm) between 1 and HSA is very close. Molecular docking studies suggested that 1 primarily binds to HSA in subdomain IIA. A protein–ligand interaction profiler was used to visualize hydrophobic, hydrogen bonds, and π–cation interactions between HSA and 1. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using HeLa and MDA-MB-231 cells showed a significant in vitro anticancer activity of 1 (IC50 2.63 and 2.68 μM, respectively). Nuclear staining assays suggested apoptotic cell death in HeLa cells treated with 1. The effect of 1 on the cytoskeletal actin filaments visualized using phalloidin staining showed extensive destruction of actin filaments. Flow cytometric analysis indicated that 1 inhibits the growth of HeLa cells through cell cycle arrest in the S phase. Western blot analysis showed upregulation in the expression of apoptotic marker proteins caspase 3, p53, and Bax. These results collectively indicate that 1 induces apoptosis by promoting DNA damage and has a high potential to act as an anticancer agent.

MutL binds to 3' resected DNA ends and blocks DNA polymerase access.

DNA mismatch repair removes mis-incorporated bases after DNA replication and reduces the error rate a 100–1000-fold. After recognition of a mismatch, a large section of up to a thousand nucleotides is removed from the daughter strand followed by re-synthesis. How these opposite activities are coordinated is poorly understood. Here we show that the Escherichia coli MutL protein binds to the 3′ end of the resected strand and blocks access of Pol I and Pol III. The cryo-EM structure of an 85-kDa MutL-DNA complex, determined to 3.7 Å resolution, reveals a unique DNA binding mode that positions MutL at the 3′ end of a primer-template, but not at a 5′ resected DNA end or a blunt DNA end. Hence, our work reveals a novel role for MutL in the final stages of mismatch repair by preventing premature DNA synthesis during removal of the mismatched strand.

Dinuclear Organoruthenium Complex for Mitochondria-Targeted Near-Infrared Imaging and Anticancer Therapy to Overcome Platinum Resistance

New mononuclear and dinuclear Ru(II) coordination compounds with the 2,7-bisbenzoimidazolyl-naphthyridine ligand have been synthesized and characterized by UV-vis, NMR, and MALDI-TOF. The molecular structures for Ru(II) compounds were determined by single-crystal X-ray diffraction. With the expansion of ligand π-conjugation and the increase in the complexed Ru number, the maximum emission wavelength red-shifted from 696 to 786 nm. The binding mode between complexes and DNA was predicted by molecular docking, which is intercalations and π-π stacking interactions with the surrounding bases. The intercalation mode of DNA binding was then determined by DNA titration and ethidium bromide (EB) displacement experiments. The antigrowth effects of complexes RuY, RuY1, and RuY2 were tested in HaCat (normal cells), HeLa (cervical cancer), A549 (lung cancer), and A549/DDP (cisplatin-resistant lung cancer) through the MTT assay. The dinuclear complex RuY2 was superior to mononuclear complexes and cisplatin in the cisplatin-resistant cell line. Confocal imaging proved that the subcellular localization of Ru(II) complexes was mitochondria; moreover, apoptosis was detected by flow cytometry. All three complexes showed a dose-dependent manner in all four cell lines. All Ru(II) complexes were found to have reactive oxygen species (ROS). The finding indicated that these Ru(II) complexes caused cell death by both DNA disruption and ROS. This study helps to explore the potential of the polynuclear Ru(II) complexes for the combination of NIR imaging and Pt-resistant cancer therapy.

Structural and functional insights into the mechanism by which MutS2 recognizes a DNA junction

MutS family proteins are classified into MutS-I and -II lineages: MutS-I recognizes mismatched DNA and initiates mismatch repair, whereas MutS-II recognizes DNA junctions to modulate recombination. MutS-I forms dimeric clamp-like structures enclosing the mismatched DNA, and its composite ATPase sites regulate DNA-binding modes. Meanwhile, the structures of MutS-II have not been determined; accordingly, it remains unknown how MutS-II recognizes DNA junctions and how nucleotides control DNA binding. Here, we solved the ligand-free and ADP-bound crystal structures of bacterial MutS2 belonging to MutS-II. MutS2 also formed a dimeric clamp-like structure with composite ATPase sites. The ADP-bound MutS2 was more flexible compared to the ligand-free form and could be more suitable for DNA entry. The inner hole of the MutS2 clamp was two times larger than that of MutS-I, and site-directed mutagenesis analyses revealed DNA-binding sites at the inner hole. Based on these, a model is proposed that describes how MutS2 recognizes DNA junctions.

Structure and DNA binding analysis of AT-rich interaction domain present in human BAF-B specific subunit BAF250b.

BAF250b and its paralog BAF250a are the DNA-binding central hub proteins present in BAF-B and BAF-A classes of SWI/SNF chromatin-remodeling complexes. BAF250b contains an AT-rich interaction domain (ARID) and C-terminal BAF250_C domain, and it is found mutated in several cancers. ARID is a conserved helix-turn-helix motif-containing DNA-binding domain present in several eukaryotic proteins. The ARID of BAF250b has been proposed to play roles in recruiting SWI/SNF to the target gene promoters for their activation. BAF250b ARID structures had been deposited in the protein data bank by a structural genomics consortium. However, it is not well-studied for its DNA-binding and solution dynamic properties. Here, we report complete backbone NMR resonance assignments of human BAF250b ARID. NMR chemical shifts and the backbone dynamics showed that the solution structure of the protein matched the reported crystal structures. The structure and chemical shift indexing revealed the presence of a short β-sheet in the DNA-binding region of BAF250b ARID that was absent in the structure of its paralog BAF250a ARID. NMR chemical shift perturbations identified DNA-binding residues and revealed the DNA-binding interface on BAF250b ARID. NMR data-driven HADDOCK models of BAF250b ARID - DNA complexes revealed its plausible mode of DNA-binding. Isothermal titration calorimetry experiments showed that BAF250b ARID interacts with DNA sequences with moderate affinities like BAF250a ARID. However, distinct thermodynamic signatures were observed for binding of BAF250a ARID and BAF250b ARID to AT-rich DNA sequence, suggesting that subtle sequence and structural differences in these two proteins influence their DNA-binding.

Xenogeneic nucleoid-associated EnrR thwarts H-NS silencing of bacterial virulence with unique DNA binding

Type III and type VI secretion systems (T3/T6SS) are encoded in horizontally acquired genomic islands (GIs) that play crucial roles in evolution and virulence in bacterial pathogens. T3/T6SS expression is subjected to tight control by the host xenogeneic silencer H-NS, but how this mechanism is counteracted remains to be illuminated. Here, we report that xenogeneic nucleoid-associated protein EnrR encoded in a GI is essential for virulence in pathogenic bacteria Edwardsiella and Salmonella. We showed that EnrR plays critical roles in T3/T6SS expression in these bacteria. Various biochemical and genetic analyses demonstrated that EnrR binds and derepresses the promoter of esrB, the critical regulator of T3/T6SS, to promote their expression by competing with H-NS. Additionally, EnrR targets AT-rich regions, globally modulates the expression of ∼363 genes and is involved in various cellular processes. Crystal structures of EnrR in complex with a specific AT-rich palindromic DNA revealed a new DNA-binding mode that involves conserved HTH-mediated interactions with the major groove and contacts of its N-terminal extension to the minor groove in the symmetry-related duplex. Collectively, these data demonstrate that EnrR is a virulence activator that can antagonize H-NS, highlighting a unique mechanism by which bacterial xenogeneic regulators recognize and regulate foreign DNA.

The many faces of the unusual biofilm activator RemA

Biofilms can be viewed as tissue-like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut-shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA-binding mode suggests that RemA organizes its target DNA into nucleosome-like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic-stress adjustment.

Defining the DNA binding mode of Dnase1L3 and modifying the enzyme to be used as a therapeutic

According to the Lupus Foundation of America, systemic lupus erythematosus (SLE) affects more than 1.5 million Americans. Common SLE treatments, including hydroxychloroquine, can cause severe side effects with long-term usage. Because of this, new treatments are needed. The SLE inflammatory response is characterized by the formation of autoantibodies against dsDNA. Nucleic acid autoantibodies can lead to the formation of immune complexes (ICs), which accumulate in the kidney and result in lupus nephritis.

Bridging the macroscopic and microscopic: a volumetric approach to DNA recognition

Living cells adapt to osmotically variable conditions in their environment. To understand the molecular basis of these adaptations, it is necessary to study the hydration properties of the biomolecular interactions that mediate the osmotic stress response. Gene expression studies have shown that the transcription factor PU.1 regulates target genes in response to cellular hyperosmotic stress, and osmotic pressure studies in vitro have shown that the high-affinity and low-affinity DNA binding modes of PU.1 are differentially hydrated.

Mixed Ni(II) and Co(II) complexes of nalidixic acid drug: Synthesis, characterization, DNA/BSA binding profile and in vitro cytotoxic evaluation against MDA-MB-231 and HepG2 cancer cell lines

In this work, herein we report the synthesis, structural characterization and in vitro cytotoxic evaluation of two mixed Co(II)/Ni(II)-nalidixic acid-bipyridyl complexes (1 and 2). The structural analysis of metal complexes 1 and 2 was carried out by analytical and multispectroscopic techniques (FT-IR, UV-vis, EPR, sXRD). The crystallographic details of complexes 1 and 2 revealed a monoclinic crystal system with P21/c space group. DFT studies of complexes were performed to get electronic structure and localization of HOMO and LUMO electron densities. Hirshfeld surface analysis of metal complexes 1 and 2 was employed to understand the various intermolecular interactions (C–H···O, N-H···H and O–H···O) that define the stability of crystal lattice structures. The comparative interaction studies of complex 1 and complex 2 with DNA/BSA were performed by diverse multispectroscopic and analytical techniques to evaluate their chemotherapeutic potential. The magnitude of the DNA binding propensity and binding mode was verified by calculating Kb, K and Ksv values. Higher binding affinity was observed in case of complex 2via intercalative mode. Furthermore, the cytotoxic assessment of complexes 1 and 2 was examined against MDA-MB-231 (triple negative human breast cancer cell line) and HepG2 (liver carcinoma cell line) employing MTT assay which revealed remarkably effecient and specific cytotoxic activity of complex 2.

Palladium(II)-quinoxaline based complexes: DNA/BSA binding, DFT, docking and anticancer activity

Substituted quinoxaline-based ligands (L1-L6) and Pd (II) complexes were synthesized. 1H/13C NMR, mass, and IR spectroscopy were used to characterize ligands and metal complexes. Structure optimization of compounds was performed using Gaussian. The newly synthesized compounds were tested for a variety of biological studies. The binding ability between complexes and CT-DNA/ protein was determined by UV–Vis spectroscopy, viscosity studies, and docking analysis; implying the intercalation mode of DNA binding. The antibacterial potency of the compounds was tested on the different types of bacteria (Gram + ve/-ve bacteria). The lower MIC of Pd(II) complexes represents the higher potency of complexes than ligands. Cytotoxicity of the produced compounds was assessed on brine shrimp. The ligands and complexes have LC50 values ranging from 7.64 to 11.45 μg/mL and 5.63–7.54 μg/mL, respectively. The cell antiproliferative nature of the compounds was checked on the MCF-7 cancer cell and the IC50 value is comparable to standard drug.