Chemical Display of Pyrimidine Bases Flipped Out by Modification-Dependent Restriction Endonucleases of MspJI and PvuRts1I Families

The epigenetic DNA modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in eukaryotes are recognized either in the context of double-stranded DNA (e.g., by the methyl-CpG binding domain of MeCP2), or in the flipped-out state (e.g., by the SRA domain of UHRF1). The SRA-like domains and the base-flipping mechanism for 5(h)mC recognition are also shared by the recently discovered prokaryotic modification-dependent endonucleases of the MspJI and PvuRts1I families. Since the mechanism of modified cytosine recognition by many potential eukaryotic and prokaryotic 5(h)mC “readers” is still unknown, a fast solution based method for the detection of extrahelical 5(h)mC would be very useful. In the present study we tested base-flipping by MspJI- and PvuRts1I-like restriction enzymes using several solution-based methods, including fluorescence measurements of the cytosine analog pyrrolocytosine and chemical modification of extrahelical pyrimidines with chloroacetaldehyde and KMnO₄. We find that only KMnO₄ proved an efficient probe for the positive display of flipped out pyrimidines, albeit the method required either non-physiological pH (4.3) or a substitution of the target cytosine with thymine. Our results imply that DNA recognition mechanism of 5(h)mC binding proteins should be tested using a combination of all available methods, as the lack of a positive signal in some assays does not exclude the base flipping mechanism.     

Comparative reactivity of mismatched and unpaired bases in relation to their type and surroundings. Chemical cleavage of DNA mismatches in mutation detection analysis

Systematic study of chemical reactivity of non-Watson–Crick base pairs depending on their type and microenvironment was performed on a model system that represents two sets of synthetic DNA duplexes with all types of mismatched and unmatched bases flanked by T·A or G·C pairs. Using comparative cleavage pattern analysis, we identified the main and additional target bases and performed quantitative study of the time course and efficacy of DNA modification caused by potassium permanganate or hydroxylamine. Potassium permanganate in combination with tetraethylammonium chloride was shown to induce DNA cleavage at all mismatched or bulged T residues, as well as at thymines of neighboring canonical pairs. Other mispaired (bulged) bases and thymine residues located on the second position from the mismatch site were not the targets for KMnO₄ attack. In contrast, hydroxylamine cleaved only heteroduplexes containing mismatched or unmatched C residues, and did not modify adjacent cytosines. However when G·C pairs flank bulged C residue, neighboring cytosines are also attacked by hydroxylamine due to defect migration. Chemical reactivity of target bases was shown to correlate strongly with the local disturbance of DNA double helix at mismatch or bulge site. With our model system, we were able to prove the absence of false-negative and false-positive results. Portion of heteroduplex reliably revealed in a mixture with corresponding homoduplex consists of 5% for bulge bases and “open” non-canonical pairs, and 10% for wobble base pairs giving minimal violations in DNA structure. This study provides a complete understanding of the principles of mutation detection methodology based on chemical cleavage of mismatches and clarifies the advantages and limitations of this approach in various biological and conformational studies of DNA.     

Boundaries of the nicking region for the F plasmid transfer origin, oriT

The extent of the F plasmid oriT nicking region was determined from the properties of successive substitution mutations in the region from base pair 121 to base pair 174 and from KMnO₄ probing of DNA structural distortions induced in vivo by tra gene products. Nicking and transfer assays indicated that the left margin of oriT Wes predominantly at the nick site, and that the nicking domain primarily lies within 17bp to the right of the nick. Some mutants that were proficient for nicking showed reduced frequencies of termination, indicating that oriT nicking does not guarantee efficient termination. DNA in the vicinity of the nick (G₁₃₇, T₁₃₈, G₁₄₀, and T₁₄₁ on the nicked strand) showed elevated sensitivity to KMnO₄ when tra gene products were present in the donor. Bases C₁₄₅, C₁₄₆, C₁₄₇, C₁₄₉, and G₁₅₀ on the un-nicked strand also became more sensitive to oxidation under tra⁺ conditions. The bases preferentially oxidized by KMnO₄ lie within the nicking domain, as defined by the substitution mutants, and they include dinucleotides that can produce kinks in the DNA. Base pairs in the nicking region are calculated to be more thermodynamically stable than base pairs in the flanking regions.     

Binding of the Bacillus subtilis LexA protein to the SOS operator

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5′-CGAACN₄GTTCG-3′. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5′ end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5′-CGAACATATGTTCG-3′. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.     

Identification of dual ligands targeting angiotensin II type 1 receptor and peroxisome proliferator-activated receptor-γ by core hopping of telmisartan

It has been reported previously that some angiotensin II receptor blockers not only antagonize angiotensin II type 1 receptor (AT₁R), but also exert stimulation in peroxisome proliferator-activated receptor γ (PPARγ) partial activation, among which telmisartan displays the best. Telmisartan has been tested as a bifunctional ligand with antihypertensive and hypoglycemic activity. Aiming at more potent leads with selective AT₁R antagonism and PPARγ partial agonism, the three parts of telmisartan including the distal benzimidazole ring, the biphenyl moiety, and the carboxylic acid group experienced modification by core hopping method in our study. The central benzimidazole ring, however, remained intact considering its great affinity toward AT₁R and PPARγ. We utilized computational techniques for the sake of details on the binding interactions and conformational stability. Standard precision docking analysis and absorption, distribution, metabolism, excretion, and toxicity prediction received 10 molecules with higher Glide scores, similar interactions, and improved pharmacokinetic profiles compared to telmisartan. Comp#91 with highest scores for AT₁R (?11.92 kcal/mol) and PPARγ (?13.88 kcal/mol) exhibited excellent binding modes and pharmacokinetic parameters. Molecular dynamics trajectories on best docking pose of comp#91 confirmed the docking results and verified the conformational stability with both receptors throughout the course of 20-ns simulations. Thus, comp#91 could be identified as a promising lead in the development of dual AT₁R antagonist and PPARγ partial agonist against hypertension and type 2 diabetes.     

Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement

The binding of PNA (peptide nucleic acid) T₂CT₂CT₄-LysNH₂ to the double-stranded DNA target 5′ -A₂GA₂GA₄ was studied by KMnO₄ and dimethylsulfate (DMS) probing. It is found that upon sequence-specific strand displacement binding of the PNA to the dsDNA target concomitant protection of the N-7 of guanines within the target takes place. It is furthermore shown that the binding of this PNA is more efficient at pH 5.5 that at pH 6.5 and very inefficient at pH 7.5. These results clearly indicate that C⁺G Hoogsteen base pairing is present and important for binding and that the strand displacement complex therefore involves a PNA·DNA-PNA triplex.     

Interaction of an anticancer benzopyrane derivative with DNA: Biophysical,biochemical, and molecular modeling studies

BackgroundSIMR1281 is a potent anticancer lead candidate with multi- target activity against several proteins; however, its mechanism of action at the molecular level is not fully understood. Revealing the mechanism and the origin of multitarget activity is important for the rational identification and optimization of multitarget drugs.MethodsWe have used a variety of biophysical (circular dichroism, isothermal titration calorimetry, viscosity, and UV DNA melting), biochemical (topoisomerase I & II assays) and computational (molecular docking and MD simulations) methods to study the interaction of SIMR1281 with duplex DNA structures.ResultsThe biophysical results revealed that SIMR1281 binds to dsDNA via an intercalation-binding mode with an average binding constant of 3.1 × 10⁶ M⁻¹. This binding mode was confirmed by the topoisomerases' inhibition assays and molecular modeling simulations, which showed the intercalation of the benzopyrane moiety between DNA base pairs, while the remaining moieties (thiazole and phenyl rings) sit in the minor groove and interact with the flanking base pairs adjacent to the intercalation site.ConclusionsThe DNA binding characteristics of SIMR1281, which can disrupt/inhibit DNA function as confirmed by the topoisomerases' inhibition assays, indicate that the observed multi-target activity might originate from ligand intervention at nucleic acids level rather than due to direct interactions with multiple biological targets at the protein level.General significanceThe findings of this study could be helpful to guide future optimization of benzopyrane-based ligands for therapeutic purposes.     

Tris (phenanthroline) Metal Complexes: Probes for DNA Helicity

Abstract

The intercalative binding of chiral tris(phenanthroline) metal complexes to DNA is stereo-selective. The enantiomeric selectivity is based upon the differential steric interactions between the two non-intercalating phenanthroline ligands of each isomer with the DNA phosphate backbone. Gel electrophoretic assays of helical unwinding, optical enrichment studies by equilibrium dialysis and luminescence titrations with separated enantiomers of (phen)₃Ru²⁺ all indicate that the delta isomer binds preferentially to the right-handed duplex. The chiral discrimination is governed by the DNA helical asymmetry. Complete stereospecifity is seen with isomers of the bulkier RuDIP (tris-4,7-diphenylphenanthrolineruthenium(II)). While both isomers bind to Z-DNA, a poor template for discrimination, binding of Λ-RuDIP to B-DNA is precluded. These chiral complexes therefore serve as a chemical probe to distinguish left and right-handed DNA helices in solution.     

Surfactant–copper(II) Schiff base complexes: synthesis,structural investigation,DNA interaction,docking studies,and cytotoxic activity

A series of surfactant–copper(II) Schiff base complexes (1–6) of the general formula, [Cu(sal-R₂)₂] and [Cu(5-OMe-sal-R₂)₂], {where, sal?=?salicylaldehyde, 5-OMe-sal?=?5-methoxy- salicylaldehyde, and R₂?=?dodecylamine (DA), tetradecylamine (TA), or cetylamine (CA)} have been synthesized and characterized by spectroscopic, ESI-MS, and elemental analysis methods. For a special reason, the structure of one of the complexes (2) was resolved by single crystal X-ray diffraction analysis and it indicates the presence of a distorted square-planar geometry in the complex. Analysis of the binding of these complexes with DNA has been carried out adapting UV-visible-, fluorescence-, as well as circular dichroism spectroscopic methods and viscosity experiments. The results indicate that the complexes bind via minor groove mode involving the hydrophobic surfactant chain. Increase in the length of the aliphatic chain of the ligands facilitates the binding. Further, molecular docking calculations have been performed to understand the nature as well as order of binding of these complexes with DNA. This docking analysis also suggested that the complexes interact with DNA through the alkyl chain present in the Schiff base ligands via the minor groove. In addition, the cytotoxic property of the surfactant–copper(II) Schiff base complexes have been studied against a breast cancer cell line. All six complexes reduced the visibility of the cells but complexes 2, 3, 5, and 6 brought about this effect at fairly low concentrations. Analyzed further, but a small percentage of cells succumbed to necrosis. Of these complexes (6) proved to be the most efficient aptotoxic agent.     

IR spectroscopic research on the impact of chemical analogues of autoregulatory d<Subscript>1</Subscript> factors of microorganisms on structural changes in DNA

Using IR spectroscopy, we investigated the impact of chemical analogues of autoregulatory d₁ factors of microorganisms (methylresorcinol, hexylresorcinol, and tyrosol) on the conformational changes in DNA in films upon altering (decreasing) the relative humidity. We analyzed the appearance/disappearance of characteristic absorption bands of A and B DNA forms and determined D ₁₀₈₈/D ₁₂₂₄, the ratio between the band intensities of symmetrical and asymmetrical oscillations in their phosphate groups. The data obtained suggest the slowing down of the B → A structural transition in DNA in the presence of methylresorcinol and its speeding up in the presence of tyrosol. We discuss the mechanisms of this phenomenon in relation to the chemical composition of d 1 factors and their biological function.     

Methacarn (methanol-Carnoy) fixation

Summary According to chemical data, methanol raises the shrinkage temperature of collagen significantly more than ethanol (86° C versus 70° C). Since increase of shrinkage temperature appears desirable in tissues to be embedded in paraffin, methanol was substituted for ethanol in Carnoy's fluid. This methanol-Carnoy mixture is referred to as methacarn solution. The fixation-embedding procedure was similar to that described in the study of Carnoy fixation. Methacarn-fixed sections showed little or no shrinkage and compared well with material fixed in Carnoy's or Zenker's fluid. Myofibrils, especially in endothelial and epithelial cells, were more prominent in methacarn- than in Carnoy-fixed tissues.A review of the chemical literature showed that methanol, ethanol and chloroform stabilize or even enhance helical conformations of proteins, presumably by strengthening of hydrogen bonds. Interference with hydrophobic bonds causes unfolding and/or structural rearrangements in globular proteins. The twin-helical structure of DNA collapses in alcoholic solutions. Hence, methacarn fixation can be expected to preserve the helical proteins in myofibrils and collagen, but the conformations of globular proteins and DNA will be significantly altered. Literature on conformational effects produced by fixatives used in electron microscopy was also reviewed. Glutaraldehyde and OsO₄ cause considerable loss of helix (22–29% and 39–66% respectively). KMnO₄ and glutaraldehyde followed by OsO₄ produce extensive transitions from helical to random-coil conformations similar to those seen in powerful denaturants such as 8 M urea. Evidently these fixatives are unsuitable for studies of helical proteins. In contrast ethylene glycol preserves helical conformations.     

Synthesis,DNA Binding,and Photonuclease Activity of New Tetraaza Macrocyclic Constrained Isoxazole Rings as Subunit in Metal Complexes

The DNA-binding and photonuclease activity of newly synthesized tetra-azamacrocyclic ligand L (C₃₂H₃₂N₈O₄) and its complexes of type [MLCl₂] and [ML]Cl₂ (where M = Co(II), Fe(II) and Cu(II); L = N,N′-[3-(4-{5-[(2-amino-ethylamino)-methyl]-isoxazol-3yl}-phenyl)-isoxazol-5-yl methyl-ethane-1,2-diamine] are specified. An octahedral geometry has been proposed for Fe(II) and Co(II) complexes, while the Cu(II) complex has a square planar environment. The absorption spectral results indicate that the complexes bind with the base pairs of DNA, with an intrinsic binding constant K_b of Fe(II), Co(II), and Cu(II) complexes found to be 3.2 × 10⁴ M^?1, 5.3 × 10⁴ M^?1, and 4.2 × 10⁴ M^?1, respectively, in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2. The large enhancement in the relative viscosity of DNA on binding to the complexes supports the proposed DNA binding modes. The viscosity and thermal denaturation studies sustain the effective intercalation with DNA. The DNA photocleavage studies demonstrated that compounds exhibit significant photonuclease activity by a concentration dependent on singlet oxygen mediated mechanism.     

Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G₊₄ base pair for the wild-type A:T₊₄ base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T₊₄ were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T₊₄ or the C:G₊₄ base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G₊₄ recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T₊₄ target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G₊₄ target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G₊₄ substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.     

Novel DNA–peptide interaction networks

Allostery in the binding of peptides to DNA has been studied by quantitative DNase I footprinting using four newly designed peptides containing the XP(Hyp)RK motif and N-methylpyrrole (Py) moieties. Apparent binding constants in the micromolar range as well as Hill coefficients were determined for each peptide. The results, together with previous studies on five other peptides support the proposal that interaction network cooperativity is highly preferred in DNA–peptide interactions that involve multiple recognition sites. It is envisaged that interstrand bidentate interactions participate in the relay of conformational changes between recognition sites on the complementary strands. Models for interpreting DNA allostery based upon interaction networks are outlined. Circular dichroism experiments involving the titration of peptides against a short oligonucleotide duplex indicate that some of these peptides bind in a dimeric manner to DNA via the minor groove, inducing characteristic conformational changes. These insights should prompt the design of new DNA-binding peptides for investigating allosteric interactions between peptides and DNA, as well as novel interaction networks, and ultimately may shed light upon the fundamental chemical rules that govern allostery in more complex biological process such as DNA–protein interaction networks.     

Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules

DNA lesions produced by aromatic isocyanates have an extra bulky group on the nucleotide bases, with the capability of forming stacking interaction within a DNA helix. In this work, we investigated the conformation of the 2′-deoxyadenosine and 2′-deoxycytidine derivatives tethering a phenyl or naphthyl group, introduced in a DNA duplex. The chemical modification experiments using KMnO₄ and 1-cyclohexyl-3 -(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate have shown that the 2′-deoxycytidine lesions form the base pair with guanine while the 2′-deoxyadenosine lesions have less ability of forming the base pair with thymine in solution. Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases. We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds. It is also presented that the attached aromatic groups change their orientation by interacting with the minor groove binding netropsin, distamycin and synthetic polyamide. The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.     

High Field 1H-NMR Analysis of the 1:1 Intercalation Complex of the Antitumor Agent Mitoxantrone and the DNA Duplex [d(CpGpCpG)]2

Abstract

Complete ¹H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]₂. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of ¹H and ¹³C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and ³¹P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

DNA binding,prominent photonuclease activity and antibacterial PDT of cobalt(II) complexes of phenanthroline based photosensitizers

Abstract

The chemistry of Co(II) complexes showing efficient light induced DNA cleavage activity, binding propensity to calf thymus DNA and antibacterial PDT is summarized in this article. Complexes of formulation [Co(mqt)(B)₂]ClO₄ 1–3 where mqt is 4-methylquinoline-2-thiol and B is N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz 3) have been prepared and characterized. The DNA-binding behaviors of these three complexes were explored by absorption spectra, viscosity measurements and thermal denaturation studies. The DNA binding constants for complexes 1, 2 and 3 were determined to be 1.6?×?10³?M^?1, 1.1?×?10⁴?M^?1 and 6.4?×?10⁴?M^?1 respectively. The experimental results suggest that these complexes interact with DNA through groove binding mode. The complexes show significant photocleavage of supercoiled (SC) DNA proceeds via a type-II process forming singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was studied using photodynamic antimicrobial chemotherapy (PACT) assay against E. coli and all complexes exhibited significant reduction in bacterial growth on photoirradiation.