DNA Conformational Changes Research Articles

Characterization of the Search Complex and Recognition Mechanism of the AlkD-DNA Glycosylase.

DNA damage is a routine problem for cells, and pathways such as base excision repair have evolved to protect the genome by using DNA glycosylases to first recognize and excise lesions. The search mechanism of these enzymes is of particular interest due to the seemingly intractable problem of probing the billions of base pairs in the genome for potential damage. It has been hypothesized that glycosylases form multiple protein-DNA conformational states to efficiently search and recognize DNA lesions, ultimately only flipping out the damaged substrate into the active site. A unique DNA glycosylase, the Bacillus cereus AlkD enzyme, has been shown to excise damaged DNA without flipping the nucleobase into a protein binding pocket following lesion recognition. Here, we use microsecond-scale all-atom molecular dynamics simulations to characterize the AlkD recognition mechanism, putting it in perspective with other DNA glycosylases. We first identify and describe two distinct enzyme-DNA conformations of AlkD: the search complex (SC) and excision complex (EC). The SC is distinguished by the linearity of DNA, changes in four helical parameters in the vicinity of the lesion, and changes in distance between active site residues and the DNA. Free DNA simulations are used to demonstrate that the DNA structural deviations and increased active site interactions present in the EC are initiated by the recognition of a methylation-induced signal in the rises both 5' to the methylation and opposing this base. Our results support the hypothesis that subtle geometric distortions in DNA are recognized by AlkD and are consequently probed to initiate concerted protein and DNA conformational changes which prime excise without additional intermediate states. This mechanism is shown to be consistent among the three methylated DNA sequences that have been crystallized bound to AlkD.

Expression Signature of Myeloma Residual Cells Is Characterized By Genes Associated with Proliferation, Epigenetic Modification, and Stem Cell Maintenance

BackgroundDespite significant progress in the treatment of multiple myeloma (MM), 30% of patients relapse within 3 years of diagnosis. Recent data indicate that intervention before relapse would benefit most patients with high-risk MM and a significant proportion (38%) of low-risk MM patients. Residual MM cells are responsible for relapse, have been described as drug resistant and as having stem cell-like characteristics including altered cell metabolism, increased drug efflux, ALDH1 activity, and myeloma propagating activity. We sought to molecularly characterize residual myeloma plasma cells (MMPCs) and identify novel biomarkers and therapeutic targets of resistant MM cells.MethodsPatient samples. Residual samples were collected at least 3 months after start of therapy (range 4 mths-11yrs), had low tumor burden (0.6-4.4% BM MMPC), 2 were in VGPR or better, 7 had PR, and 3 had not responded. Of the 12 cases analyzed, 2 had relapsed > 1 yr prior to residual sample.RNAseq analysis. Paired MMPCs at diagnosis and during residual disease (n=12) and 4 healthy donors were interrogated by RNAseq. Genes with at least 2-fold difference between groups and at a statistical significance <0.05 were investigated further. String pathway analysis was used to detect pathways and biological functions enriched in resistant myeloma cells. The drug gene interaction database (DGIdb) was used to identify known and potential drug targets.ResultsRNAseq shows that paired diagnostic and residual samples cluster together. Of genes differentially expressed in residual disease, there was reduced expression of cell adhesion markers (e.g. CXCR1, CXCR2, CEACAM3, CEACAM8, ITGB3, ITGAM), tumor suppressors (e.g. PPARG, TP53I11) and metabolic genes (e.g. HK3, PYGL) and increased expression of genes associated with proliferation (e.g. KIF11, OIP5, MAD2L1, BIRC5, ZWINT, MKI67), chromosomal instability (e.g. TOP2A, KIF4A, PBK, RRM2), epigenetic modification (e.g. EZH2, DNMT3B, HMGN5), high centrosome index (e.g. TYMS, MAD2L1, TOP2A, BIRC5, ZWINT, PLK4), DNA repair (e.g. FANCI, FANCB, RAD51, MND1, BRCA1) and stem cell activity such as Notch signaling, ALDH1 expression, and drug efflux (e.g. DLL4, KIF14, TOP2A, BIRC5). We identified cell surface markers EPHA5, TRPC4, MAGEA1, MAGEA12, GAGE1, NLGN1, SVIL, CCR9, KCNK2, PLK4, CIT, and DMD that will be tested for their ability to identify resistant MMPCs in future studies. Interrogation of the DGIdb for drugs targeting genes upregulated in residual MMPCs revealed 36 targets with known drugs available and an additional 23 targets that are potentially druggable (i.e. cell surface, kinase, transporter, DNA repair etc.).To better understand the mechanisms involved in maintaining residual disease, genes that were exclusively upregulated in residual cells were entered into the STRING bioinformatics platform for pathway analysis. This revealed an interaction network of 186 nodes (genes) and 599 edges (molecular interactions), with the average node degree (the number of edges connected to the node) being 6.44. The clustering coefficient and protein-protein interaction enrichment P-value were 0.374 and 1x10-16, respectively.The genes expressed in residual MMPCs were enriched in cell cycle pathways including, chromosome segregation, nuclear division, mitotic cell cycle, positive regulation of cell cycle, cell division, DNA conformation change, DNA metabolic process, DNA packaging, and chromosome organization (FDR < 1%).ConclusionsWe have identified a gene expression signature specific to resistant myeloma which may be used for early detection of relapse and development of novel therapeutic targets. Interestingly, many genes associated with high risk in myeloma were expressed in residual samples despite the majority of cases being molecularly-defined low risk at diagnosis (11/12), suggesting that residual myeloma harbors a high risk expression signature without gain of genomic aberrations. Identifying markers of resistance early in the disease course will allow treatments to be modified to prevent relapse and may reduce long-term exposure to chemotherapeutics. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Flap Endonuclease 1 Mutations A159V and E160D Cause Genomic Instability by Slowing Reaction on Double-Flap Substrates

Flap endonuclease 1 (FEN1) is a structure-selective nuclease best known for its roles in the penultimate steps of Okazaki fragment maturation, long-patch base excision repair and ribonucleotide excision repair. To better understand the role of FEN1 in genome maintenance in yeast and mammals, FEN1 active site mutations (A159V and E160D) have been used as tools to dissect its involvement in DNA metabolic pathways. However, discrepancies concerning the biochemistry and molecular etiology of genomic instability when FEN1 function is altered exist. Here, a detailed biochemical and biophysical characterization of mouse FEN1 and mutants is presented. Kinetic measurements showed that the active site mutants A159V and E160D reduce the rates of hydrolysis under multiple- and single-turnover conditions on all substrates. Consistent with their dominant negative effects in heterozygotes, neither mutation affects the adoption of the substrate duplex arms in the bent conformation on the enzyme surface, although decreases in substrate binding affinity are observed. The ability of the mutants to induce the requisite local DNA conformational change near the scissile phosphate is adversely affected, suggesting that the ability to place the scissile phosphate optimally in the active site causes the reduction in rates of phosphate diester hydrolysis. Further analysis suggests that the A159V mutation causes the chemistry of phosphate diester hydrolysis to become rate-limiting, whereas the wild-type and E160D proteins are likely rate-limited by a conformational change. On the basis of these results, the proposed roles of FEN1 in genome maintenance derived from studies involving these mutations are reassessed.

MutSγ-Induced DNA Conformational Changes Provide Insights into Its Role in Meiotic Recombination

In many organisms, MutSγ plays a role in meiotic recombination, facilitating crossover formation between homologous chromosomes. Failure to form crossovers leads to improper segregation of chromosomes and aneuploidy, which in humans result in infertility and birth defects. To improve current understanding of MutSγ function, this study investigates the binding affinities and structures of MutSγ in complex with DNA substrates that model homologous recombination intermediates. For these studies, we overexpressed and isolated from Escherichia coli the yeast MutSγ protein Saccharomyces cerevisiae (Sc) Msh4-Msh5. Sc Msh4-Msh5 binds Holliday junction (HJ)-like substrates, 3′ overhangs, single-stranded (ss) forks, and the displacement loop with nanomolar affinity. The weakest binding affinities are detected for an intact duplex and open-junction construct. Similar to the human protein, Sc Msh4-Msh5 exhibits the highest affinity for the HJ with a Kd < 0.4 nM in solution. Energy-transfer experiments further demonstrate that DNA structure is modulated by the binding interaction with the largest changes associated with substrates containing an ss end. Upon binding, Sc Msh4-Msh5 displaces the ss away from the duplex in most of the ss-containing intermediates, potentially enabling the binding of RPA and other proteins. In the case of the junction-like intermediates, Msh4-Msh5 binding either stabilizes the existing stacked structure or induces formation of the stacked X conformation. Significantly, we find that upon binding, Msh4-Msh5 stacks an open-junction construct to the same extent as the standard junction. Stabilization of the junction in the stacked conformation is generally refractory to branch migration, which is consistent with a potential role for MutSγ to stabilize HJs and prevent branch migration until resolution by MutLγ. The different binding modalities observed suggest that Msh4-Msh5 not only binds to and stabilizes stacked junctions but also participates in meiotic recombination before junction formation through the stabilization of single-end invasion intermediates.

Analysis of DNA-Protein Complexes by Atomic Force Microscopy Imaging: The Case of TRF2-Telomeric DNA Wrapping.

Atomic force microscopy (AFM) is a non-optical microscopy that enables the acquisition at the nanoscale level of a 3D topographical image of the sample. For 30years, AFM has been a valuable tool in life sciences to study biological samples in the field of tissue, cellular and molecular imaging, of mechanical properties and of force spectroscopy. Since the early beginnings of the technique, AFM has been extensively exploited as an imaging tool for structural studies of nucleic acids and nucleoprotein complexes. The morphometric analysis performed on the images can unveil specific structural and functional aspects of the sample, such as the multimerization state of proteins bound to DNA, or DNA conformational changes led by the DNA-binding proteins. Herein, a method for analyzing a complex formed by a telomeric DNA sequence wrapped around the TRF2 binding protein is presented. The described procedure could be applied to the study of any type of DNA-protein complex.

Substrate specificity of human apurinic/apyrimidinic endonuclease APE1 in the nucleotide incision repair pathway.

Human apurinic/apyrimidinic (AP) endonuclease APE1 catalyses the hydrolysis of phosphodiester bonds on the 5′ side of an AP-site (in the base excision repair pathway) and of some damaged nucleotides (in the nucleotide incision repair pathway). The range of substrate specificity includes structurally unrelated damaged nucleotides. Here, to examine the mechanism of broad substrate specificity of APE1, we performed pulsed electron–electron double resonance (PELDOR) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer (FRET) detection of DNA conformational changes during DNA binding and lesion recognition. Equilibrium PELDOR and kinetic FRET data revealed that DNA binding by APE1 leads to noticeable damage-dependent bending of a DNA duplex. Molecular dynamics simulations showed that the damaged nucleotide is everted from the DNA helix and placed into the enzyme’s binding pocket, which is formed by Asn-174, Asn-212, Asn-229, Ala-230, Phe-266 and Trp-280. Nevertheless, no damage-specific contacts were detected between these amino acid residues in the active site of the enzyme and model damaged substrates containing 1,N6-ethenoadenosine, α-adenosine, 5,6-dihydrouridine or F-site. These data suggest that the substrate specificity of APE1 is controlled by the ability of a damaged nucleotide to flip out from the DNA duplex in response to an enzyme-induced DNA distortion.

Coordinated protein and DNA conformational changes govern mismatch repair initiation by MutS

MutS homologs identify base-pairing errors made in DNA during replication and initiate their repair. In the presence of adenosine triphosphate, MutS induces DNA bending upon mismatch recognition and subsequently undergoes conformational transitions that promote its interaction with MutL to signal repair. In the absence of MutL, these transitions lead to formation of a MutS mobile clamp that can move along the DNA. Previous single-molecule FRET (smFRET) studies characterized the dynamics of MutS DNA-binding domains during these transitions. Here, we use protein–DNA and DNA–DNA smFRET to monitor DNA conformational changes, and we use kinetic analyses to correlate DNA and protein conformational changes to one another and to the steps on the pathway to mobile clamp formation. The results reveal multiple sequential structural changes in both MutS and DNA, and they suggest that DNA dynamics play a critical role in the formation of the MutS mobile clamp. Taking these findings together with data from our previous studies, we propose a unified model of coordinated MutS and DNA conformational changes wherein initiation of mismatch repair is governed by a balance of DNA bending/unbending energetics and MutS conformational changes coupled to its nucleotide binding properties.

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase.

Some DNA transposons relocate from one genomic location to another using a mechanism that involves generating double-strand breaks at their transposon ends by forming hairpins on flanking DNA. The same double-strand break mode is employed by the V(D)J recombinase at signal-end/coding-end junctions during the generation of antibody diversity. How flanking hairpins are formed during DNA transposition has remained elusive. Here, we describe several co-crystal structures of the Hermes transposase bound to DNA that mimics the reaction step immediately prior to hairpin formation. Our results reveal a large DNA conformational change between the initial cleavage step and subsequent hairpin formation that changes which strand is acted upon by a single active site. We observed that two factors affect the conformational change: the complement of divalent metal ions bound by the catalytically essential DDE residues, and the identity of the –2 flanking base pair. Our data also provides a mechanistic link between the efficiency of hairpin formation (an A:T basepair is favored at the –2 position) and Hermes' strong target site preference. Furthermore, we have established that the histidine residue within a conserved C/DxxH motif present in many transposase families interacts directly with the scissile phosphate, suggesting a crucial role in catalysis.

Real-time label-free analysis of the thermostability of DNA structures using GelRed

In biological systems, conformational transformations of nucleic acids play critical roles in genetic regulation. However, it remains a tricky task to design and optimize specific labeling strategies to track these changes. In this study, we exploited an intercalating fluorescent dye, GelRed, to characterize different DNA structures. We studied the correlation between fluorescence intensity and DNA structural properties. We showed that single-stranded DNAs with predicted self-folded secondary structures show much stronger fluorescence than those without such structures. For double-stranded DNAs, we observed that fluorescence intensity is positively correlated to their GC content. We also demonstrated that GelRed can be used to monitor DNA conformational changes upon temperature variations in real time. Based on these findings, we concluded that the fluorescence intensity of a GelRed-stained DNA structure has a good correlation with its thermostability in the form of a change in Gibbs free energy.

Reduction of DNA Folding by Ionic Liquids and Its Effects on the Analysis of DNA-Protein Interaction Using Solid-State Nanopore.

DNA folding is not desirable for solid-state nanopore techniques when analyzing the interaction of a biomolecule with its specific binding sites on DNA since the signal derived from the binding site could be buried by a large signal from the folding of DNA nearby. To resolve the problems associated with DNA folding, ionic liquids (ILs), which are known to interact with DNA through charge-charge and hydrophobic interactions are employed. 1-n-butyl-3-methylimidazolium chloride (C4 mim) is found to be the most effective in lowering the incident of DNA folding during its translocation through solid-state nanopores (4-5 nm diameter). The rate of folding signals from the translocation of DNA-C4 mim is decreased by half in comparison to that from the control bare DNA. The conformational changes of DNA upon complexation with C4 mim are further examined using atomic force microscopy, showing that the entanglement of DNA which is common in bare DNA is not observed when treated with C4 mim. The stretching effect of C4 mim on DNA strands improves the detection accuracy of nanopore for identifying the location of zinc finger protein bound to its specific binding site in DNA by lowering the incident of DNA folding.

Sensitive detection of oxidative DNA damage in cyanobacterial cells using supercoiling-sensitive quantitative PCR

Supercoiling-sensitive quantitative PCR (ss-qPCR) is a sensitive technique to detect DNA damage in cultured animal cells and cultured/clinical human cells in vitro. In this study, we investigated whether the ss-qPCR method can be applied as a sensitive means to detect oxidative DNA damage in unicellular organisms. We used the model cyanobacterium Synechococcus elongatus PCC 7942 as a test organism and H2O2 as an exogenetic oxidative toxicant. Results showed that a significant increase in the plasmid DNA damage of S. elongatus PCC 7942 was induced by H2O2 in a dose- and time-dependent manner. The sensitivity of ss-qPCR in detecting DNA damage of the cyanobacterium was higher than the cell inhibition method (up to 255 times) as calculated from the slopes of fitted curves in the tested sub-toxic concentration range of 1–5 mM H2O2. Ss-qPCR also detected repairable low-intensity DNA damage in the cyanobacterium when DNA repair inhibitors were used. The detection limit of modified ss-qPCR was one tenth of that of previous methods. We also observed that ss-qPCR can be used to detect genomic DNA conformation change of cyanobacterium exposed to H2O2. Thus, this method will provide a powerful technical support for investigating the mechanisms of cyanobacterial DNA damage by environmental factors, especially intracellular reactive oxygen species enhancement-related factors.

Evaluation of Anticancer Activity of Extracted Flavonoids from Morus Alba Leaves and its interaction with DNA

ABSTRACT Morous Alba, known as white mulberry contains many oxidative flavonoids, widely used in the treatment of many diseases like hyperglycemia, inflammation, fever and cancer. In the present study we investigated the interaction of extracted flavonoids from Iranian Morus Alba leaves with DNA as a main target for anticancer drugs. Various spectroscopic techniques (UV/Vis, CD and Fluorescence Spectroscopy) were used to detect the interaction. In vivo studies also were done to confirm the effectiveness of the extracted flavonoids. The spectroscopic results showed that the extracted flavonoids bind to DNA especially to the sugar-phosphate backbone and making DNA conformational changes upon this binding. Experiments on the cancerous mice with solid tumors indicated that the treatment of mice with these extracted flavonoids increased significantly the life span but they did not have any effects on the tumor size reduction. These data suggest that Morus Alba flavonoids may use as an effective natural anticancer drug in the near future.

Nanoscale Investigation into the Cellular Response of Glioblastoma Cells Exposed to Protons.

Exposure to ionizing radiation can induce cellular defense mechanisms including cell activation and rapid proliferation prior to metastasis and in extreme cases can result in cell death. Herewith we apply infrared nano- and microspectroscopy combined with multidimensional data analysis to characterize the effect of ionizing radiation on single glioblastoma nuclei isolated from cells treated with 10 Gy of X-rays or 1 and 10 Gy of protons. We observed chromatin fragmentation related to the formation of apoptotic bodies following X-ray exposure. Following proton irradiation we detected evidence of a DNA conformational change (B-DNA to A-DNA transition) related to DNA repair and accompanied by an increase in protein content related to the synthesis of peptide enzymes involved in DNA repair. We also show that proton exposure can increase cholesterol and sterol ester synthesis, which are important lipids involved in the metastatic process changing the fluidity of the cellular membrane in preparation for rapid proliferation.

Two-stage DNA compaction induced by silver ions suggests a cooperative binding mechanism.

The interaction between silver ions and DNA plays an important role in the therapeutic use of silver ions and in related technologies such as DNA sensors. However, the underlying mechanism has not been fully understood. In this study, the dynamics of Ag+-DNA interaction at a single-molecule level was studied using magnetic tweezers. AgNO3 solutions with concentrations ranging from 1 μM to 20 μM led to a 1.4-1.8 μm decrease in length of a single λ-DNA molecule, indicating that Ag+ has a strong binding with DNA, causing the DNA conformational change. The compaction process comprises one linear declining stage and another sigmoid-shaped stage, which can be attributed to the interaction mechanism. Considering the cooperative effect, the sigmoid trend was well explained using a phenomenological model. By contrast, addition of silver nanoparticle solution induced no detectable transition of DNA. The dependence of the interaction on ionic strength and DNA concentration was examined via morphology characterization and particle size distribution measurement. The size of the Ag+-DNA complex decreased with an increase in Ag+ ionic strength ranging from 1 μM to 1 mM. Morphology characterization confirmed that silver ions induced DNA to adopt a compacted globular conformation. At a fixed [AgNO3]:[DNA base pairs] ratio, increasing DNA concentration led to increased sizes of the complexes. Intermolecular interaction is believed to affect the Ag+-DNA complex formation to a large extent.

The Cucurbit[7]Uril-Based Supramolecular Chemistry for Reversible B/Z-DNA Transition.

As a left‐handed helical structure, Z‐DNA is biologically active and it may be correlated with transcription and genome stability. Until recently, it remained a significant challenge to control the B/Z‐DNA transition under physiological conditions. The current study represents the first to reversibly control B/Z‐DNA transition using cucurbit[7]uril‐based supramolecular approach. It is demonstrated that cucurbit[7]uril can encapsulate the central butanediamine moiety [HN(CH2)4NH] and reverses Z‐DNA caused by spermine back to B‐DNA. The subsequent treatment with 1‐adamantanamine disassembles the cucurbit[7]uril/spermine complex and readily induces reconversion of B‐ into Z‐DNA. The DNA conformational change is unequivocally demonstrated using different independent methods. Direct evidence for supramolecular interactions involved in DNA conformational changes is further provided. These findings can therefore open a new route to control DNA helical structure in a reversible way.

Cooperative recognition of T:T mismatch by echinomycin causes structural distortions in DNA duplex.

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.

Sensitive and label-free electrochemical lead ion biosensor based on a DNAzyme triggered G-quadruplex/hemin conformation

Lead ion (Pb2+) is a common environmental contaminant, which causes serious bioaccumulation and toxicity in human body. In this work, we developed a novel Pb2+ electrochemical biosensor using the specific DNAzyme on a DNA tetrahedron probe, in the presence of Pb2+, the substrate strand was cleaved into two parts and released a “G-rich” oligo which subsequently formed a G-quadruplex/hemin complex, generating a detectable catalysis current signal with the assistant of H2O2. The 3-D DNA tetrahedron regulated the density and orientation of the probe and thus improved the DNAzyme reaction, and facilitated the complex DNA conformational change in the confined space of the interface on the electrode surface, Finally, the LOD of our biosensor was proved to be 0.008 nM (3σ), which is 9000 times lower than the safety limit of EPA (15 μg/L or 72 nM), and 6000 times lower than IARC (10 μg/L or 48.26 nM), and more importantly, the specificity and reproducibility of the proposed biosensor was well demonstrated.

Compaction of Single-Molecule Megabase-Long Chromatin under the Influence of Macromolecular Crowding

The megabase-sized length of chromatin is highly relevant to the state of chromatin in vivo, where it is subject to a highly crowded environment and is organized in topologically associating domains of similar dimension. We developed an in vitro experimental chromatin model system reconstituted from T4 DNA (approximately 166 kbp) and histone octamers and studied the monomolecular compaction of this megabase-sized chromatin fiber under the influence of macromolecular crowding. We used single-molecule fluorescence microscopy and observed compaction in aqueous solutions containing poly(ethylene glycol) in the presence of monovalent (Na+ and K+) and divalent (Mg2+) cations. Both DNA and chromatin demonstrated compaction under comparable conditions in the presence of poly(ethylene glycol) and Na+ or Mg2+ salt. However, the mechanism of the compaction changed from a first-order phase transition for DNA to a continuous folding for megabase-sized chromatin fibers. A more efficient and pronounced chromatin compaction was observed in the presence of Na+ compared to K+. A flow-stretching technique to unfold DNA and chromatin coils was used to gain further insight into the morphology of partially folded chromatin fibers. The results revealed a distribution of partially folded chromatin fibers. This variability is likely the result of the heterogeneous distribution of nucleosomes on the DNA chain. The packaging of DNA in the form of chromatin in the crowded nuclear environment appears essential to ensure gradual conformational changes of DNA.

FoldX accurate structural protein-DNA binding prediction using PADA1 (Protein Assisted DNA Assembly 1).

The speed at which new genomes are being sequenced highlights the need for genome-wide methods capable of predicting protein–DNA interactions. Here, we present PADA1, a generic algorithm that accurately models structural complexes and predicts the DNA-binding regions of resolved protein structures. PADA1 relies on a library of protein and double-stranded DNA fragment pairs obtained from a training set of 2103 DNA–protein complexes. It includes a fast statistical force field computed from atom-atom distances, to evaluate and filter the 3D docking models. Using published benchmark validation sets and 212 DNA–protein structures published after 2016 we predicted the DNA-binding regions with an RMSD of <1.8 Å per residue in >95% of the cases. We show that the quality of the docked templates is compatible with FoldX protein design tool suite to identify the crystallized DNA molecule sequence as the most energetically favorable in 80% of the cases. We highlighted the biological potential of PADA1 by reconstituting DNA and protein conformational changes upon protein mutagenesis of a meganuclease and its variants, and by predicting DNA-binding regions and nucleotide sequences in proteins crystallized without DNA. These results opens up new perspectives for the engineering of DNA–protein interfaces.

INTERACTION OF TWO FLAVONOIDS WITH CALF THYMUS DNA: A MULTI - SPECTROSCOPIC, ELECTROCHEMICAL AND MOLECULAR MODELLING APPROACH

Interaction of naturally occurring bioactive flavonoids 5,6,7-trihydroxyflavone (Baicalein) and 7,8-dihydroxyflavone (DHF) binding with calf thymus deoxyribose nucleic acid (dsDNA) was studied by employing UV absorption, fluorescence, circular dichroism, cyclic voltammetric and molecular modeling techniques. All studies were confirmed that the structural changes of DNA binding to the flavonoid. From the CV results positive shift in peak potential and increased peak current of the flavonoid in the presence of DNA and then the fluorescence quenching of DNA-flavonoids system indicated the intercalative mode of binding between flavonoid and DNA. CD studies suggest the conformational changes in DNA upon interaction with the flavonoids. Molecular docking simulation methods are used as tools to delineate the binding mode and probable location of the flavonoids and their effects on the stability and conformation of Ct-(ds) DNA. Furthermore, Baicalein can bind with more potential with Ct-(ds) DNA than DHF. This is helpful to understand the molecular aspects of binding mode and provides direction for the use and the design of new effective therapeutic agents. These results could provide useful information for insight into the pharmacological mechanism of flavonoids.