Selective DNA Binding Research Articles

Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition

The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future.

DNA Condensing Effects and Sequence Selectivity of DNA Binding of Antitumor Noncovalent Polynuclear Platinum Complexes

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

Thioester Bonds of Thiocoraline Can Be Replaced with NMe-Amide Bridges without Affecting Its DNA-Binding Properties.

In the search for new drug candidates for DNA recognition, affinity and sequence selectivity are two of the most important features. NMe-azathiocoraline, a synthetic antitumor bisintercalator derived from the natural marine product thiocoraline, shows similar potency to the parent compound, as well as possessing enhanced stability. Analysis of the DNA-binding selectivity of NMe-azathiocoraline by DNase I footprinting using universal substrates with all 136 tetranucleotides and all possible symmetrical hexanucleotide sequences revealed that, although this ligand binds to all CpG steps with lower affinities than thiocoraline, it displays additional binding to AT-rich sites. Moreover, fluorescence melting studies showed a strong interaction of the synthetic molecule with CACGTG and weaker binding to ACATGT and AGATCT. These findings demonstrate that NMe-azathiocoraline has the same mode of action as thiocoraline, mimicking its DNA-binding selectivity despite the substitution of its thioester bonds by NMe-amide bridges.

Dual topoisomerase I and II poisoning by chiral Ru(II) complexes containing 2-thiophenylimidazo[4,5-f][1,10]phenanthroline derivatives

A series of chiral Ru(II) complexes bearing thiophene ligands were synthesized and characterized. Both Ru(II) complexes Δ/Λ-[Ru(bpy)2(pscl)]2+ (Δ/Λ-1) and Δ/Λ-[Ru(bpy)2(psbr)]2+ (Δ/Λ-2) (bpy=2,2′-bipyridine, pscl=2-(5-chlorothiophen-2-yl)imidazo[4,5-f][1,10]phenanthroline, psbr=2-(5-bromothiophen-2-yl)imidazo[4,5-f][1,10]phenanthroline) showed antitumor activities against A549, HepG2 and BEL-7402 tumor cell lines, especially HeLa tumor cell line. Moreover, Δ enantiomers were more active than Λ enantiomers, accounting for the different cellular uptake. In addition, with the extension of time, these enantiomers could finally accumulate in the nucleus, suggesting that nucleic acids were the cellular target of these enantiomers. The DNA-binding behaviors of complexes were studied using spectroscopic and viscosity measurements. Results suggested that four complexes could bind to DNA in an intercalative mode but no obvious DNA-binding selectivity between the enantiomers was observed. Topoisomerase inhibition and DNA religation assay confirmed that four complexes acted as efficient dual topoisomerase I and II poisons, DNA strand breaks had also been observed from alkaline single cell gel electrophoresis (comet assay). Δ-1 and Δ-2 inhibited the growth of HeLa cells through the induction of apoptotic cell death, as evidenced by the Alexa Fluor® 488 annexin V staining assays and flow cytometry analysis. The results demonstrated that Δ/Λ-1 and Δ/Λ-2 acted as dual topoisomerase I and II poisons and caused DNA damage that could lead to cell cycle arrest by apoptosis.

Biorecognition by DNA Oligonucleotides after Exposure to Photoresists and Resist Removers

Combining biological molecules with integrated circuit technology is of considerable interest for next generation sensors and biomedical devices. Current lithographic microfabrication methods, however, were developed for compatibility with silicon technology rather than bioorganic molecules, and consequently it cannot be assumed that biomolecules will remain attached and intact during on-chip processing. Here, we evaluate the effects of three common photoresists (Microposit S1800 series, PMGI SF6, and Megaposit SPR 3012) and two photoresist removers (acetone and 1165 remover) on the ability of surface-immobilized DNA oligonucleotides to selectively recognize their reverse-complementary sequence. Two common DNA immobilization methods were compared: adsorption of 5'-thiolated sequences directly to gold nanowires and covalent attachment of 5'-thiolated sequences to surface amines on silica coated nanowires. We found that acetone had deleterious effects on selective hybridization as compared to 1165 remover, presumably due to incomplete resist removal. Use of the PMGI photoresist, which involves a high temperature bake step, was detrimental to the later performance of nanowire-bound DNA in hybridization assays, especially for DNA attached via thiol adsorption. The other three photoresists did not substantially degrade DNA binding capacity or selectivity for complementary DNA sequences. To determine whether the lithographic steps caused more subtle damage, we also tested oligonucleotides containing a single base mismatch. Finally, a two-step photolithographic process was developed and used in combination with dielectrophoretic nanowire assembly to produce an array of doubly contacted, electrically isolated individual nanowire components on a chip. Postfabrication fluorescence imaging indicated that nanowire-bound DNA was present and able to selectively bind complementary strands.

Protein recognition of the S23906-1–DNA adduct by nuclear proteins: direct involvement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

In a view to develop new DNA alkylating antitumour drugs, evaluating the precise mechanism of action and the molecular/cellular consequences of the alkylation is a point of major interest. The benzo-b-acronycine derivative S23906-1 alkylates guanine nucleobases in the minor groove of the DNA helix and presents an original ability to locally open the double helix of DNA, which appears to be associated with its cytotoxic activity. However, the molecular mechanism linking adduct formation to cellular consequences is not precisely known. The objective of the present study was to identify proteins involved in the recognition and mechanism of action of S23906-DNA adducts. We found that GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a protein that binds to S23906-alkylated single-stranded, double-stranded and telomeric sequences in a drug-dependent and DNA sequence/structure-dependent manner. We used the CASTing (cyclic amplification of sequence targeting) method to identify GAPDH DNA-binding selectivity and then evaluated its binding to such selected S23906-alkylated sequences. At the cellular level, alkylation of S23906-1 results in an increase in the binding of GAPDH and its protein partner HMG (high-mobility group) B1 to the chromatin. Regarding the multiple roles of GAPDH in apoptosis and DNA repair, the cytotoxic and apoptotic activities of GAPDH were evaluated and present opposite effects in two different cellular models.

Effect of Flanking Bases on the DNA Specificity of EmBP-1

EmBP-1 is a basic region leucine zipper (bZIP) protein found in many types of plants. In general, plant bZIP proteins bind selectively to DNA sequences containing ACGT core sequences with different immediate flanking nucleotides preferred by different proteins. I report that the distant flanking sequence also has a strong effect on the preference of EmBP-1 for internal bases and determine the residue governing this effect. EmBP-1 binds selectively to the 10 bp gcG-box palindrome GCCACGTGGC 18-fold more tightly than the gcC-box GTGACGTCAC, but when the outer flanking G/C residues were changed to A/T (i.e., ACCACGTGGT and ATGACGTCAT), an only 1.2-fold preference for G-box binding was observed. Analysis of a series of single-residue alanine mutants of EmBP-1 revealed that this effect is mediated by arginine 10. Mutation of this residue to alanine greatly reduces the affinity for the gcG-box while leaving the affinity for other sequences relatively unchanged. Partial retention of G-box specificity upon mutation of R10 to lysine indicates that the effect is reliant on the basic nature of the residue. Additional studies with other EmBP-1 protein mutants and with oligonucleotides containing the T/A and C/G flanking sequences demonstrate the complexity of the protein-DNA interaction and demonstrate that the mechanism of sequence selective DNA binding is highly dependent on the flanking sequence.

Metallohelices with activity against cisplatin-resistant cancer cells; does the mechanism involve DNA binding?

Enantiomers of a relatively rigid DNA-binding metallo-helix are shown to have comparable activity to that of cisplatin against the cell lines MCF7 (human breast adenocarcinoma) and A2780 (human ovarian carcinoma) but are ca five times more active against the cisplatin-resistant A2780cis. The cell-line HCT116 p53+/+ (human colon carcinoma) is highly sensitive giving IC50 values in the nM range, far lower than the cisplatin control. The hypothesis that the biological target of such metallohelices is DNA is probed by various techniques. Tertiary structure changes in ct-DNA (formation of loops and intramolecular coiling) on exposure to the compounds are demonstrated by atomic force microscopy and supported by circular/linear dichroism in solution. Selectivity for 5′-CACATA and 5′-CACTAT segments is shown by DNase I footprinting. Various three- and four-way oligonucleotide junctions are stabilised, and remarkably only the Λ metallo-helix enantiomer stabilizes T-shaped 3WJs during gel electrophoresis; this is despite the lack of a known helix binding site. In studies with oligonucleotide duplexes with bulges it is also shown for the first time that the metallo-helix binding strength and the number of binding sites are dependent on the size of the bulge. In contrast to all the above, flexible metallo-helices show little propensity for structured or selective DNA binding, and while for A2780 the cancer cell line cytotoxicity is retained the A2780cis strain shows significant resistance. For all compounds in the study, H2AX FACS assays on HCT116 p53+/+ showed that no significant DNA damage occurs. In contrast, cell cycle analysis shows that the DNA binders arrest cells in the G2/mitosis phase, and while all compounds cause apoptosis, the DNA binders have the greater effect. Taken together these screening and mechanistic results are consistent with the more rigid helices acting via a DNA binding mechanism while the flexible assemblies do not.

Ni2+ and Cu2+ complexes of a phenanthroline-based ligand bind to G-quadruplexes at non-overlapping sites

Transition metal complexes allow fine tuning of DNA binding affinity and selectivity. Here we report on the nucleic acid recognition properties of a phenanthroline-based ligand coordinated to Ni(2+) or Cu(2+). The resulting complexes clearly bind to telomeric G-quadruplexes at different sites according to the nature of the bound metal ion.

Ternary copper(ii)-polypyridyl enantiomers: aldol-type condensation, characterization, DNA-binding recognition, BSA-binding and anticancer property

Chiral enantiomers [Cu(phen)(L-threo)(H2O)]NO3 1 and [Cu(phen)(D-threo)(H2O)]NO3 2 (threo = threoninate) underwent aldol-type condensation with formaldehyde, with retention of chirality, to yield their respective enantiomeric ternary copper(II) complexes, viz. L- and D-[Cu(phen)(5MeOCA)(H2O)]NO3·xH2O (3 and 4; phen = 1,10-phenanthroline; 5MeOCA = 5-methyloxazolidine-4-carboxylate; x = 0-3) respectively. These chiral complexes were characterized by FTIR, elemental analysis, circular dichroism, UV-Visible spectroscopy, fluorescence spectroscopy (FL), molar conductivity measurement, ESI-MS and X-ray crystallography. Analysis of restriction enzyme inhibition by these four complexes revealed modulation of DNA binding selectivity by the type of ligand, ligand modification and chirality. Their interaction with bovine serum albumin was investigated by FL and electronic spectroscopy. With the aid of the crystal structure of BSA, spectroscopic evidence suggested their binding at the cavity containing Trp134 with numerous Tyr residues in subdomain IA. The products were more antiproliferative than cisplatin against cancer cell lines HK-1, MCF-7, HCT116, HSC-2 and C666-1 except HL-60, and were selective towards nasopharyngeal cancer HK-1 cells over normal NP69 cells of the same organ type.

Biological activity and DNA binding studies of 2-substituted benzimidazo[1,2-a]quinolines bearing different amino side chains

This manuscript describes the synthesis and biological activity of 2-substituted benzimidazo[1,2-a]quinolines substituted with different amino side chains on the quinoline nucleus prepared by microwave assisted amination. The majority of compounds were newly synthesized and active at submicromolar IC50 concentrations, while the alkylamino substituents, either acyclic or cyclic, increased antitumor activities in comparison with previously published nitro and amino substituted benzimidazo[1,2-a]quinolines. The compound with the longest tertiary amino side chain (16) was the least active. A series of additional experiments, including DNA binding propensities, topoisomerases I and II inhibition, inhibition of recombinant green fluorescent protein in a cell-free translation system, cell cycle perturbances and cellular localization, was performed to shed more light on the mechanisms of action of the most active compounds. The DNA intercalation activity correlates with anti-proliferative effect. Several DNA intercalators (11, 20 and 21) also evidence some sequence selective DNA binding. However, only N,N-dimethylaminopropyl analogue 11 was unequivocally demonstrated to be a strong DNA-binder and intercalative agent, which efficiently targets DNA in the cells, while the activity of compound 10, with a bulky i-butylamino side chain, points to its potential antimitotic activity.

Correlation of Local Effects of DNA Sequence and Position of β-Alanine Inserts with Polyamide–DNA Complex Binding Affinities and Kinetics

To improve our understanding of the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔC(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.

Promoter scanning of the human COX-2 gene with 8-ring polyamides: Unexpected weakening of polyamide–DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine

Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal β-alanines as polyamide building blocks in place of N-methylpyrrole; β-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with β-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with β-alanine to generate a β/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that β-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.

Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli

The Escherichia coli very short patch (VSP) repair pathway corrects thymidine-guanine mismatches that result from spontaneous hydrolytic deamination damage of 5-methyl cytosine. The VSP repair pathway requires the Vsr endonuclease, DNA polymerase I, a DNA ligase, MutS, and MutL to function at peak efficiency. The biochemical roles of most of these proteins in the VSP repair pathway have been studied extensively. However, these proteins have not been studied together in the context of VSP repair in an in vitro system. Using purified components of the VSP repair system in a reconstitution reaction, we have begun to develop an understanding of the role played by each of these proteins in the VSP repair pathway and have gained insights into their interactions. In this report we demonstrate an in vitro reconstitution of the VSP repair pathway using a plasmid DNA substrate. Surprisingly, the repair track length can be modulated by the concentration of DNA ligase. We propose roles for MutL and MutS in coordination of this repair pathway.

Spi-1/PU.1 activates transcription through clustered DNA occupancy in erythroleukemia

Acute leukemias are characterized by deregulation of transcriptional networks that control the lineage specificity of gene expression. The aberrant overexpression of the Spi-1/PU.1 transcription factor leads to erythroleukemia. To determine how Spi-1 mechanistically influences the transcriptional program, we combined a ChIP-seq analysis with transcriptional profiling in cells from an erythroleukemic mouse model. We show that Spi-1 displays a selective DNA-binding that does not often cause transcriptional modulation. We report that Spi-1 controls transcriptional activation and repression partially through distinct Spi-1 recruitment to chromatin. We revealed several parameters impacting on Spi-1-mediated transcriptional activation. Gene activation is facilitated by Spi-1 occupancy close to transcriptional starting site of genes devoid of CGIs. Moreover, in those regions Spi-1 acts by binding to multiple motifs tightly clustered and with similar orientation. Finally, in contrast to the myeloid and lymphoid B cells in which Spi-1 exerts a physiological activity, in the erythroleukemic cells, lineage-specific cooperating factors do not play a prevalent role in Spi-1-mediated transcriptional activation. Thus, our work describes a new mechanism of gene activation through clustered site occupancy of Spi-1 particularly relevant in regard to the strong expression of Spi-1 in the erythroleukemic cells.

γ sulphate PNA (PNA S): Highly Selective DNA Binding Molecule Showing Promising Antigene Activity

Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.

DNA-Capped Mesoporous Silica Nanoparticles as an Ion-Responsive Release System to Determine the Presence of Mercury in Aqueous Solutions

We have developed DNA-functionalized silica nanoparticles for the rapid, sensitive, and selective detection of mercuric ion (Hg(2+)) in aqueous solution. Two DNA strands were designed to cap the pore of dye-trapped silica nanoparticles. In the presence of ppb level Hg(2+), the two DNA strands are dehybridized to uncap the pore, releasing the dye cargo with detectable enhancements of fluorescence signal. This method enables rapid (less than 20 min) and sensitive (limit of detection, LOD, 4 ppb) detection, and it was also able to discriminate Hg(2+) from twelve other environmentally relevant metal ions. The superior properties of the as-designed DNA-functionalized silica nanoparticles can be attributed to the large loading capacity and highly ordered pore structure of mesoporous silica nanoparticles, as well as the selective binding of thymine-rich DNA with Hg(2+) . Our design serves as a new prototype for metal-ion sensing systems, and it also has promising potential for detection of various targets in stimulus-release systems.

Flow-Through Purification of Viruses- A Novel Approach to Vaccine Purification

A two step flow-through chromatography process is proposed as an universal approach to purify viruses. A resin column with reduced surface area was developed for the first step to remove bulk of the host cell protein (HCP) from a viral feed stream while allowing most of the virus to flow-through. For the second step a chromatographic separations strategy using a primary amine membrane adsorber and multivalent ions in the mobile phase was developed. This enabled selective binding of host cell DNA (hcDNA) to the membrane and complete recovery of virus in the flow-through mode. The techniques were evaluated using cell culture grown influenza virus and bacteriophage feed streams. Virus recoveries of >70-80% and 100% were achieved for the column and membrane approaches respectively. The column cleared > 80% of the HCP and the membrane adsorber reduced whole hcDNA levels to <10 ng.

Phosphopeptides Designed for 5-Methylcytosine Recognition

An artificial phosphopeptide has been developed through rational design of the interaction with 5-methylcytosine in duplex DNA. The peptide consists of two tandem zinc finger motifs, in one of which the glutamate was replaced with a phosphotyrosine, the phosphotyrosine in the peptide being effective for methylcytosine selectivity of DNA binding. The flexible modulation of the target methylated sequence by rearrangement of zinc finger peptides is possible, and the phosphopeptide provided us an important hint for expansion of the codes for the interactions of zinc fingers with DNA to methylated DNA sequences. The fluorescence-labeled phosphopeptide provided information on the methylation status of genomic DNA through fluorescence anisotropy after a 10 min incubation.

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA+) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA+ domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA+ domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.