(dA·dT)-dependent inactivation of the DNA template properties by interaction with netropsin and distamycin A

The inhibitory effect of the polypeptide antibiotics netropsin and distamycin A on DNA dependent nucleic acid synthesis has been shown to be related to the base composition of the template DNA. A number of natural DNA's of quite different dA·dT content as well as poly (dI-dC)·poly (dI-dC), poly (dA-dT)·poly (dA-dT), poly (dA) · poly (dT) and poly (dG)·poly(dC) has been studied as templates in DNA and in part in RNA polymerase reaction. The highest binding efficiency of netropsin existing for (dA·dT)-containing DNA polymers and the less pronounced interaction with the (dI·dC)-containing polymer shown by the melting and CD spectral behaviour of the complexes are entirely reflected in the template inactivation. The same is evident for distamycin A. However, in contrast to netropsin the antibiotic distamycin A exhibits some binding tendency to poly (dG)·poly (dC). Binding effects of a netropsin derivative to DNA and (dA·dT)-containing polymers suggest the importance of hydrogen bonds of the peptide groups in the complex formation.     

Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K_m^AdoMet, for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 µM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K_m^DNA values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 µM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT ≥ CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-l-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.     

Structure and Dynamics of Netropsin-Poly(dA-dT)· Poly(dA-dT) Complex: 500 MHz 1H NMR Studies

Abstract

Antibiotic netropsin is known to bind specifically to A and T regions in DNA; the mode of binding being non-intercalative. Obviously, H-bonding between the proton donors of netropsin and acceptors N3 of A and 02 of T comes as a strong possibility which might render this specificity. In netropsin there could be 8 proton donors: four terminal amino groups and four internal imino groups. However, methylation of the terminal amino groups does not alter the binding affinity of netropsin to DNA—but the modification of the internal imino groups significantly lowers the binding affinity. Hence, the logical conclusion is that netropsin may specifically interact with A and T through H-bonding and in order to do so, it should approach the helix from the minor groove. The present paper provides experimental data which verify the conclusion mentioned above.

Using poly(dA-dT)? poly(dA-dT) as a model system it was observed following a thorough theoretical stereochemical analysis that netropsin could bind to -(T-A-T) sequence of the polymer in the B-form through the minor groove by forming specific B-bonding. Models could be either right or left-handed B-DNA with a mono or dinucleotide repeat.

By monitoring the ³¹P signals of free poly(dA-dT) ? poly(dA-dT) and netropsin-poly(dA-dT)? poly(dA-dT) complex we show that the drug changes the DNA structure from essentially a mononucleotide repeat to that of very dominant dinucleotide repeat; however the base- pairing in the DNA-drug complex remain to be Watson-Crick. Whether H-bonding is the specific mode of interaction was judged by monitoring the imino protons of netropsin in the presence of poly(dA-dT) ? poly(dA-dT). This experiment was conducted in 90% H₂O + 10% D₂O Using the time-shared long pulse. It was found that exchangeable imino protons of netropsin appear in the drug-DNA complex and disappear upon increasing the D₂O content; thus confirming that H-bonding is indeed the specific mode of interaction. From these and several NOE measurements, we propose a structure for poly(dA-dT)? poly(dA-dT(-netropsin complex.

In summary, experimental data indicate that netropsin binds to poly(dA-dT)? poly(dA-dT) by forming specific hydrogen bonds and that the binding interaction causes the structure to adopt a Watson-Crick paired dinucleotide repeat motif. The proposed hydrogen bonds can form only if the drug approaches the DNA from the minor groove. Within the NMR time scale the interaction between the ligand and DNA is a fast one. From the NOE experimental data, it appears that poly(dA-dT)? poly(dA-dT) in presence of netropsin exists as an equilibrium mixture of right- and left-handed B-DNA duplexes with a dinucleotide repeat—with a predominance of the left-handed form. The last conclusion is a soft one because it was very difficult to make sure the absence of spin diffusion. In a 400 base pairs long DNA duplex- drug complex (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis.     

Structural analysis of DNA complexation with cationic lipids

Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid–DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K_Chol = 1.4 (±0.5) × 10⁴ M⁻¹, K_DDAB = 2.4 (±0.80) × 10⁴ M⁻¹, K_DOTAP = 3.1 (±0.90) × 10⁴ M⁻¹ and K_DOPE = 1.45 (± 0.60) × 10⁴ M⁻¹. The order of stability of lipid–DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content.     

Entropically-driven binding of mithramycin in the minor groove of C/G-rich DNA sequences

The antitumour antibiotic mithramycin A (MTA) is a DNA minor-groove binding ligand. It binds to C/G-rich tracts as a dimer that forms in the presence of divalent cations such as Mg²⁺. Differential scanning calorimetry, UV thermal denaturation, isothermal titration calorimetry and competition dialysis were used, together with computations of the hydrophobic free energy of binding, to determine the thermodynamic profile of MTA binding to DNA. The results were compared to those obtained in parallel using the structurally related mithramycin SK (MSK). The binding of MTA to salmon testes DNA determined by UV melting studies (K_obs = 1.2 (±0.3) × 10⁵M⁻¹) is tighter than that of MSK (2.9 (±1.0) × 10⁴M⁻¹) at 25°C. Competition dialysis studies showed a tighter MTA binding to both salmon testes DNA (42% C + G) and Micrococcus lysodeikticus DNA (72% C + G). The thermodynamic analysis of binding data at 25°C shows that the binding of MTA and MSK to DNA is entropically driven, dominated by the hydrophobic transfer of the antibiotics from solution to the DNA-binding site. Direct molecular recognition between MTA or MSK and DNA through hydrogen bonding and van der Waals contacts may also contribute significantly to complex formation.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

Interaction of Sulforaphane with DNA and RNA

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO₂), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO₂) interaction. Overall binding constants were estimated to be K_(SFN–DNA)=3.01 (± 0.035)×10⁴ M^-1 and K_(SFN–RNA)= 6.63 (±0.042)×10³ M^-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Two Binding Modes of Netropsin are Involved in the Complex Formation with Poly(dA-dT)·Poly(dA-dT) and other Alternating DNA Duplex Polymers

Abstract

Using CD measurements we show that the interaction of netropsin to poly(dA-dT)·poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA·dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA)·poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT)·poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.     

Ultrasound-mediated DNA transfer for bacteria

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5α and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl₂, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 × 10⁹ CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 × 10⁻⁶ transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5α and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 × 10⁻⁶ and 4.33 ± 0.78 × 10⁻⁶ transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.     

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.     

Molecular dynamics simulations and free energy calculations of netropsin and distamycin binding to an AAAAA DNA binding site

Molecular dynamics simulations have been performed on netropsin in two different charge states and on distamycin binding to the minor groove of the DNA duplex d(CGCGAAAAACGCG)·d(CGCGTTTTTCGCG). The relative free energy of binding of the two non-covalently interacting ligands was calculated using the thermodynamic integration method and reflects the experimental result. From 2 ns simulations of the ligands free in solution and when bound to DNA, the mobility and the hydrogen-bonding patterns of the ligands were studied, as well as their hydration. It is shown that even though distamycin is less hydrated than netropsin, the loss of ligand–solvent interactions is very similar for both ligands. The relative mobilities of the ligands in their bound and free forms indicate a larger entropic penalty for distamycin when binding to the minor groove compared with netropsin, partially explaining the lower binding affinity of the distamycin molecule. The detailed structural and energetic insights obtained from the molecular dynamics simulations allow for a better understanding of the factors determining ligand–DNA binding.     

Equilibrium and kinetic studies on the binding of des-N-tetramethyltriostin A to DNA

The interaction between TANDEM (a des-methyl analogue of triostin A) and poly(dA-dT) results in extension of the helix by 6.8 Å for each ligand molecule bound, exactly as predicted for a bis-intercalation reaction. Cooperativity is evident in Scatchard plots for the interaction at ionic strengths of 0.2 and 1.0, where the binding constant is diminished compared to that which pertains at low salt concentration. Binding to a natural DNA (calf thymus), already considerably weaker than binding to poly(dA-dT), is also sensitive to increased ionic strength. With a self-complementary octanucleotide d(G-G-T-A-T-A-C-C) the binding curve indicates the presence of a single des-N-tetramethyltriostin A binding site per helical fragment with a non-cooperative association constant about 6·10⁶ M^?1. Detergent-induced dissociation of des-N-tetramethyltriostin A-poly(dA-dT) complexes results in a simple exponential decay at all levels of binding, but the time constant of decay is dependent upon the initial binding ratio. This behaviour cannot directly explain the cooperativity of equilibrium binding isotherms but suggests the occurrence of relatively long-lived perturbations of the helical structure by binding of the ligand. [Ala³, Ala⁷]des-N-tetramethyltriostin A, which has a more flexible octapeptide ring lacking the disulphide cross-bridge, dissociates from poly(dA-dT) much faster than des-N-tetramethyltriostin A. Dissociation of des-N-tetramethyltriostin A from calf thymus DNA is more rapid than dissociation of triostin A or other quinoxaline antibiotics, which may account for its low antimicrobial activity.     

Molecular recognition of poly(A) by small ligands: an alternative method of analysis reveals nanomolar, cooperative and shape-selective binding

A few drug-like molecules have recently been found to bind poly(A) and induce a stable secondary structure (T_m ≈ 60°C), even though this RNA homopolymer is single-stranded in the absence of a ligand. Here, we report results from experiments specifically designed to explore the association of small molecules with poly(A). We demonstrate that coralyne, the first small molecule discovered to bind poly(dA), binds with unexpectedly high affinity (K_a >10⁷ M⁻¹), and that the crescent shape of coralyne appears necessary for poly(A) binding. We also show that the binding of similar ligands to poly(A) can be highly cooperative. For one particular ligand, at least six ligand molecules are required to stabilize the poly(A) self-structure at room temperature. This highly cooperative binding produces very sharp transitions between unstructured and structured poly(A) as a function of ligand concentration. Given the fact that junctions between Watson–Crick and A·A duplexes are tolerated, we propose that poly(A) sequence elements and appropriate ligands could be used to reversibly drive transitions in DNA and RNA-based molecular structures by simply diluting/concentrating a sample about the poly(A)-ligand ‘critical concentration’. The ligands described here may also find biological or medicinal applications, owing to the 3′-polyadenylation of mRNA in living cells.     

Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR

We have performed solid-state ³¹P-¹⁹F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA^2′FUTGGC)·d(GCCAAT(pS)TT GCG) (A₃T₂) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the ^2′FU7–pS19 inter-label distance, was 9.4 ± 0.7 Å, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 Å decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 Å, consistent with the results of a previous solution NMR study of the 2:1 complex. These ³¹P-¹⁹F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7–14 Å in DNA–drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.     

Characterization of a Baculovirus-Encoded ATP-Dependent DNA Ligase

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [α-³²P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) · poly(dT)_12–18, or poly(rA) · poly(dT)_12–18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.     

A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase

Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3′-azido-2′,3′-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5′-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s⁻¹) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 µM) and chemistry forward and reverse reactions, 0.38 and 0.22 s⁻¹, respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing k_cat/K_m (7 × 10⁻⁶ μ M⁻¹ s⁻¹). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5′-triphosphate (8-oxo-dGTP) show this same phenomena.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Controlling nucleic acid secondary structure by intercalation: effects of DNA strand length on coralyne-driven duplex disproportionation

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)·poly(dT) into triplex poly(dA)·poly(dT)·poly(dT) and a poly(dA) self- structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)₁₆·(dT)₁₆ by coralyne reverts over the course of hours if the sample is maintained at 4°C. Coralyne-disproportioned (dA)₃₂· (dT)₃₂, on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)₁₆·(dT)₁₆ sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)₁₆ self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)_n self-structure] binds coralyne in a length-dependent manner.