The influence of polyamine-nucleic acid complexes on Fe2+ autoxidation

Summary Polyamines are able to affect Fe²⁺ autoxidation in the presence of suitable low molecular weight phosphorus-containing compounds; the inhibitory effect exerted by polyamines is directly related to their ability to bind phosphorus-containing compounds [1].It is well known that polyamines, as polycations at physiological pH, bind strongly to nucleic acids. In this paper it is shown that polyamines, also in the presence of nucleic acids, inhibit Fe²⁺ autoxidation and thus depress the generation of free oxygen radicals. Most of the nucleic acids tested inhibited Fe²⁺ autoxidation although the concentration which causes half maximal effect differs. Polyamine effect on Fe²⁺ autoxidation varies greatly depending on the single or double stranded nature of the nucleic acid. In the present of single stranded nucleic acids, spermine and spermidine potentiate the inhibition of Fez+ autoxidation by these nucleic acids. A relationship exists between the ability of spermine to interact with single stranded nucleic acids and to inhibit Fe²⁺ autoxidation in their presence. When double stranded nucleic acids are present, polyamines reverse the inhibition of Fee+ autoxidation exerted by these nucleic acids. Molecular mechanisms are proposed to explain these experimental results. The hypothesis that polyamines may inhibit oxidative damage caused to nucleic acids by Fe²⁺ autoxidation, is also discussed.Abbreviations poly [A] polyadenylic acid (5) - poly [C] polycytidylic acid (5) - poly [1] polyinosinic acid (5) - poly [G] polyguanylic acid (5) - poly [A. U] polyadenylic-uridylic acid - poly [A] poly [U] polyadenylic-polyuridylic acid     

Interactions of polyamines with the DNA octamers d(m5CG)4 and d(GGAATTCC): A 1H‐NMR investigation

The interaction of two DNA octamers, d(m⁵CG)₄ and d(GGAATTCC), with the polyamines spermine⁴⁺ and spermidine³⁺, has been studied by means of ¹H‐nmr nuclear Overhauser effect (NOE) difference measurements. The experiments were performed at 10°C and for a polyamine charge to DNA charge (i.e., phosphate) ratio of 0.4, where the solution of d(m⁵CG)₄ contains about 50% Z‐form of the DNA. The results show that the polyamine intramolecular NOEs for the protons on the propyl chains are similarly negative with the two oligonucleotides, while those on the butyl chain show slightly more negative NOE with d(m⁵CG)₄ than with d(GGAATTCC). The fully N‐methylated analogues of spermine (Me₁₀Spn⁴⁺) and spermidine (Me₈Spd³⁺) as well as the diamines 1,3‐diaminopropane (DAP²⁺) and 1,4‐diaminobutane (putrescine²⁺) have been studied for the ability to transform d(m⁵CG)₄ from the B‐ to the Z‐form. ¹H‐nmr spectra showed the order spermine⁴⁺ > spermidine³⁺ > Me₁₀Spn⁴⁺ > Me₈Spd³⁺ > 1,3‐diaminopropane²⁺ > putrescine²⁺, with spermine showing the largest relative amount of Z‐DNA. ¹H‐nmr pulsed‐gradient self‐diffusion measurements of the triamines showed a large difference in the interaction of Spd and Me₈Spd with the two different duplexes. With the same duplex (either of the two), however, no difference between Spd and Me₈Spd can be seen. Within a two‐state model this is interpreted as a larger fraction of bound polyamines with d(m⁵CG)₄ than with d(GGAATTCC). © 1999 John Wiley & Sons, Inc. Biopoly 49: 41–53, 1999     

Molecular dynamics simulation study of oriented polyamine- and Na-DNA: sequence specific interactions and effects on DNA structure

Molecular dynamics (MD) computer simulations have been carried out on four systems that correspond to an infinite array of parallel ordered B-DNA, mimicking the state in oriented DNA fibers and also being relevant for crystals of B-DNA oligonucleotides. The systems were all comprised of a periodical hexagonal cell with three identical DNA decamers, 15 water molecules per nucleotide, and counterions balancing the DNA charges. The sequence of the double helical DNA decamer was d(5'-ATGCAGTCAG)xd(5'-TGACTGCATC). The counterions were the two natural polyamines spermidine(3+) (Spd(3+)) and putrescine(2+) (Put(2+)), the synthetic polyamine diaminopropane(2+) (DAP(2+)), and the simple monovalent cation Na(+). This work compares the specific structures of the polyamine- and Na-DNA systems and how they are affected by counterion interactions. It also describes sequence-specific hydration and interaction of the cations with DNA. The local DNA structure is dependent on the nature of the counterion. Even the very similar polyamines, Put(2+) and DAP(2+), show clear differences in binding to DNA and in effect on hydration and local structure. Generally, the polyamines disorder the hydration of the DNA around their binding sites whereas Na(+) being bound to DNA attracts and organizes water in its vicinity. Cation binding at the selected sites in the minor and in the major groove is compared for the different polyamines and Na(+). We conclude that the synthetic polyamine (DAP(2+)) binds specifically to several structural and sequence-specific motifs on B-DNA, unlike the natural polyamines, Spd(3+) and Put(2+). This specificity of DAP(2+) compared to the more dynamic behavior of Spd(3+) and Put(2+) may explain why the latter polyamines are naturally occurring in cells.     

Methylated analogs of spermine and spermidine as tools to investigate cellular functions of polyamines and enzymes of their metabolism

Biogenic amines spermine (Spm) and spermidine (Spd) are essential for cell growth. Polyamine analogs are widely used to investigate the enzymes of polyamine metabolism and the functions of spermine and spermidine in vitro and in vivo. It was demonstrated recently that α-methylated derivatives of Spm and Spd are able to fulfill the key cellular functions of polyamines, moreover, in some cases, the effects of (R) and (S) isomers were actually different. Using these α-methylated analogs of Spm and Spd, it turned possible to prevent the development of acute pancreatitis in SSAT-transgenic rats with controllable expression of the Spm/Spd N¹-acetyltransferase gene. The analogs made it possible to reveal dormant stereospecificity of polyamine oxidase, Spm oxidase, and deoxyhypusine synthase. An original approach was suggested to regulate the stereospecificity of polyamine oxidase. Depletion of the intracellular polyamine pool was found to have both hypusine-related consequences and consequences unrelated to posttranslational modification of the eukaryotic translation initiation factor eIF5A. Possible applications of a new family of C-methylated polyamine analogs for the investigation and regulation of polyamine metabolism in vitro and in vivo are discussed.     

Influence of some chromophore substituents on the intercalation of anthracycline antibiotics into DNA

The interaction between some chromopore-modified daunorubicin derivatives and calf thymus DNA was studied using a number of physical techniques in order to investigate the effect substituents on the aromatic ring system have on the capacity to intercalate into DNA and on the DNA binding affinity. The modifications examined include methylation of the hydroxyl groups at the 6 and 11 positions of the B ring and removal of the 11-hydroxyl group. The studies showed that only 11-deoxydaunorubicin retains the ability to bind to DNA by the intercalation mechanism typical of the parent compound, although the structural modification leads to an appreciably weaker binding. In contrast, methylation of any hydroxyl group dramatically reduces the affinity of the drug for DNA. At physiological ionic strength both methyl ether derivatives showed no evidence of intercalation. Structure activity correlations for the intercalation reaction deduced from these studies are in agreement with earlier findings and hypotheses relating to antitumour activity.     

2,4-Dichlorophenoxy acetic acid and indoleacetic acid partially counteract inhibition of organogenesis by difluoromethylornithine

Difluoromethylornithine (DFMO) is a well known inhibitor of putrescine biosynthesis that has been reported to interact in varying ways with auxins such as indoleacetic acid (IAA) and 2,4-dichlorophenoxy acetic acid (2,4-D). In the present report DFMO is shown to inhibit root formation in isolated hypocotyl segments of Euphorbia esula L. (leafy spurge) grown in the dark on solidified nutrient media in Petri dishes. Shoot formation was only slightly inhibited by DFMO and only on media with salts and vitamins diluted 10-fold. 2,4-D inhibited both root and shoot formation in full strength or diluted media. Simultaneous application of both compounds resulted in partial reversal of root inhibition, but only at 450 n M 2,4-D, the highest concentration used. In both media IAA also partially reversed DFMO effects on root formation. The effects of DFMO, 2,4-D or IAA on root (or shoot) formation do not appear to be closely related to endogenous content of the polyamines determined by high performance liquid chromatography.     

Protein-nucleic acid recognition: statistical analysis of atomic interactions and influence of DNA structure

We analyzed structural features of 11,038 direct atomic contacts (either electrostatic, H-bonds, hydrophobic, or other van der Waals interactions) extracted from 139 protein-DNA and 49 protein-RNA nonhomologous complexes from the Protein Data Bank (PDB). Globally, H-bonds are the most frequent interactions (approximately 50%), followed by van der Waals, hydrophobic, and electrostatic interactions. From the protein viewpoint, hydrophilic amino acids are over-represented in the interaction databases: Positively charged amino acids mainly contact nucleic acid phosphate groups but can also interact with base edges. From the nucleotide point of view, DNA and RNA behave differently: Most protein-DNA interactions involve phosphate atoms, while protein-RNA interactions involve more frequently base edge and ribose atoms. The increased participation of DNA phosphate involves H-bonds rather than salt bridges. A statistical analysis was performed to find the occurrence of amino acid-nucleotide pairs most different from chance. These pairs were analyzed individually. Finally, we studied the conformation of DNA in the interaction sites. Despite the prevalence of B-DNA in the database, our results suggest that A-DNA is favored in the interaction sites.     

Investigating the kinetics of DNA–DNA and PNA–DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy

Plasmon surface polaritons, resonantly excited in the Kretschmann format, are used to enhance the fluorescence emission of chromophore-labeled oligonucleotides (15mers) binding to surface-attached (via biotin–streptavidin linkages) complement catcher probes. A detailed analysis of the association and dissociation kinetics as well as the affinity constants is given for a mismatch 1 hybrid, emphasizing, in particular, the experimental conditions that are required to allow for an artifact-free determination of rate constants. A first comparison between DNA- and peptide nucleic acid (PNA-) probes shows similar affinities, however, significant deviations from single-exponential kinetics predicted by a simple Langmuir model for the PNA case are found.     

Manipulation of the polyamine content and senescence of apical buds of G2 peas

The effect of various treatments on the apical senescence and polyamine content of apical buds of G2 peas was analysed. Defruiting prevented senescence and increased bud size and polyamine content. Exogenous applications of GA₂₀ enhanced bud size and spermidine concentration. Applied spermidine had a slight effect on spermidine level but did not delay senescence. ACC strongly induced adecrease in bud size and, at 10 mM, apical senescence. This was accompanied by a steady decline in the level of all polyamines though their concentration remained constant until 10 mM ACC, where a drop was noted. Spermidine in the presence of ACC modulated the effect of ACC on the bud size while returning the internal polyamine content to control levels. AVG, an inhibitor of ACC synthesis produced pronounced increases in putrescine though no apparent effect on apical bud growth. Polyamine synthesis inhibitors were without effect on growth or internal polyamine content. The internal polyamine content appeared to correlate with apical bud size and vigor but did not show any consistent relationship to apical bud senescence.     

Crystal structures of A-DNA duplexes

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G · T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G · ^m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G · C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology. © 1997 John Wiley & Sons, Inc. Biopoly 44: 45–63, 1997     

Criticality of a conserved tyrosine residue in the SpeG protein from Escherichia coli

The SpeG spermidine/spermine N‐acetyltransferase (SSAT) from Escherichia coli belongs to the Gcn5‐related N‐acetyltransferase (GNAT) superfamily of proteins. In vitro characterization of this enzyme shows it acetylates the polyamines spermine and spermidine, with a preference toward spermine. This enzyme has a conserved tyrosine residue (Y135) that is found in all SSAT proteins and many GNAT functional subfamilies. It is located near acetyl coenzyme A in the active center of these proteins and has been suggested to act as a general acid in a general acid/base chemical mechanism. In contrast, a previous study showed this residue was not critical for E. coli SpeG enzymatic activity when mutated to phenylalanine. This result was quite different from previous studies with a comparable residue in the human and mouse SSAT proteins, which also acetylate spermine and spermidine. Therefore, we constructed several mutants of the E. coli SpeG Y135 residue and tested their enzymatic activity. We found this conserved residue was indeed critical for E. coli SpeG enzyme activity and may behave similarly in other SSAT proteins.     

Recognition of muramic acid and N-acetylmuramic acid by Leguminosae lectins: possible role in plant-bacteria interactions

Abstract Interaction of 24 different seed lectins/ isolectins from the Leguminosae with muramic acid (MurAc), N-acetylmuramic acid (MurNAc) and muramyl-dipeptides (MDP), was studied by hapten-inhibition of haemagglutination. Although many lectins were shown to interact, irrespective of their monosaccharide-specificity or systematic position, glucose/mannose-specific lectins from the tribe Vicieae exhibited the best affinity for these components of the bacterial cell wall. The discrepancies observed in the binding of the muramyl-dipeptide diastereo-isomers to lectins suggest that the binding is somewhat conformation-dependent. These interactions could be possibly involved in the recognition of bacteria by plants.     

草地螟滞育幼虫的蛋白和核酸含量变化

为了阐明草地螟Loxostege sticticalis 滞育的分子调控基础, 本研究应用Trizol法、 DNA和蛋白质定量试剂盒、 SDS-PAGE电泳技术分别对进入滞育1, 2, 3和4个月、 解除滞育以及非滞育草地螟老熟幼虫中的核酸含量、 总蛋白含量和组分的变化进行了测定。结果表明： 滞育不同月份幼虫的总RNA含量显著低于非滞育幼虫的总RNA含量（P＜0.05）; 解除滞育幼虫的总RNA含量显著高于滞育2, 3和4个月的幼虫, 但仍显著低于非滞育的对照组。滞育不同月份、 非滞育以及解除滞育幼虫的总DNA含量没有显著差异（P＞0.05）。RNA/DNA比值随滞育的开始而显著下降, 随着滞育的结束而显著上升。滞育不同月份的幼虫总蛋白含量显著高于非滞育幼虫的总蛋白含量（P<0.05）, 但解除滞育与非滞育幼虫的总蛋白含量无显著差异。利用SDS-PAGE电泳分析发现滞育幼虫体内存在滞育关联蛋白, 蛋白条带在24 kDa左右, 但非滞育和已经解除滞育的幼虫则没有该蛋白条带。这些结果表明, 总RNA含量的降低、 RNA/DNA比值下降、 总蛋白含量的升高, 以及24 kDa蛋白的存在是草地螟幼虫滞育的主要生理特征。     

qNABpredict: Quick,accurate, and taxonomy-aware sequence-based prediction of content of nucleic acid binding amino acids

Protein sequence-based predictors of nucleic acid (NA)-binding include methods that predict NA-binding proteins and NA-binding residues. The residue-level tools produce more details but suffer high computational cost since they must predict every amino acid in the input sequence and rely on multiple sequence alignments. We propose an alternative approach that predicts content (fraction) of the NA-binding residues, offering more information than the protein-level prediction and much shorter runtime than the residue-level tools. Our first-of-its-kind content predictor, qNABpredict, relies on a small, rationally designed and fast-to-compute feature set that represents relevant characteristics extracted from the input sequence and a well-parametrized support vector regression model. We provide two versions of qNABpredict, a taxonomy-agnostic model that can be used for proteins of unknown taxonomic origin and more accurate taxonomy-aware models that are tailored to specific taxonomic kingdoms: archaea, bacteria, eukaryota, and viruses. Empirical tests on a low-similarity test dataset show that qNABpredict is 100 times faster and generates statistically more accurate content predictions when compared to the content extracted from results produced by the residue-level predictors. We also show that qNABpredict's content predictions can be used to improve results generated by the residue-level predictors. We release qNABpredict as a convenient webserver and source code at http://biomine.cs.vcu.edu/servers/qNABpredict/ . This new tool should be particularly useful to predict details of protein–NA interactions for large protein families and proteomes.     

Conformation and packing properties of phosphatidic acid: The crystal structure of monosodium dimyristoylphosphatidate

The conformation and molecular packing of monosodium 1,2-dimyristoyl-sn-glycerophosphate (DMPA) has been determined by single crystal analysis (R = 0.107). The lipid crystallizes in the space group P2₁ with unit cell dimensions: $\text{a = 5.44, b = 7.95, c = 43.98}\text{A}\text{?}$ and β = 114.2°. The two molecules of the unit cell are related by a two-fold screw axis and pack tail-to-tail in a bilayer structure. The monosodium phosphate group packs with rather a small cross-section (24 Ų) relative to the two hydrocarbon chains. This unbalance in packing cross-section is overcome by an interdigitation of the phosphate head groups of adjacent bilayers and the formation of a single, common phosphate group layer at the bilayer interfaces. The phosphate groups are linked by hydrogen bonds to linear strands which laterally are separated by strands of sodium ions. The conformation of the molecules differs from that of other phospholipids. The glycerol chain is oriented parallel (instead of perpendicular) to the layer surface and the parallel stacking of the hydrocarbon chains is achieved by a bend of the γ-chain (instead of the β-chain). Otherwise the conformation of the glycerol dicarboxyl ester group displays the same preferred features as generally found in glycerophospholipids. The hydrocarbon chains pack according to the triclinic (T_∥) packing mode. The interaction and packing principles of the phosphate head group are discussed in relation to the structural behaviour of phosphatidic acid.     

Crystal structure of the novel PaiA N-acetyltransferase from Thermoplasma acidophilum involved in the negative control of sporulation and degradative enzyme production

GCN5-related N-acetyltransferases (GNATs) are the most widely distributed acetyltransferase systems among all three domains of life. GNATs appear to be involved in several key processes, including microbial antibiotic resistance, compacting eukaryotic DNA, controlling gene expression, and protein synthesis. Here, we report the crystal structure of a putative GNAT Ta0374 from Thermoplasma acidophilum, a hyperacidophilic bacterium, that has been determined in an apo-form, in complex with its natural ligand (acetyl coenzyme A), and in complex with a product of reaction (coenzyme A) obtained by cocrystallization with spermidine. Sequence and structural analysis reveals that Ta0374 belongs to a novel protein family, PaiA, involved in the negative control of sporulation and degradative enzyme production. The crystal structure of Ta0374 confirms that it binds acetyl coenzyme A in a way similar to other GNATs and is capable of acetylating spermidine. Based on structural and docking analysis, it is expected that Glu53 and Tyr93 are key residues for recognizing spermidine. Additionally, we find that the purification His-Tag in the apo-form structure of Ta0374 prevents binding of acetyl coenzyme A in the crystal, though not in solution, and affects a chain-flip rotation of "motif A" which is the most conserved sequence among canonical acetyltransferases.     

The role of molecular structure of sugar‐phosphate backbone and nucleic acid bases in the formation of single‐stranded and double‐stranded DNA structures

Our previous DFT computations of deoxydinucleoside monophosphate complexes with Na⁺‐ions (dDMPs) have demonstrated that the main characteristics of Watson‐Crick (WC) right‐handed duplex families are predefined in the local energy minima of dDMPs. In this work, we study the mechanisms of contribution of chemically monotonous sugar‐phosphate backbone and the bases into the double helix irregularity. Geometry optimization of sugar‐phosphate backbone produces energy minima matching the WC DNA conformations. Studying the conformational variability of dDMPs in response to sequence permutation, we found that simple replacement of bases in the previously fully optimized dDMPs, e.g. by constructing Pyr‐Pur from Pur‐Pyr, and Pur‐Pyr from Pyr‐Pur sequences, while retaining the backbone geometry, automatically produces the mutual base position characteristic of the target sequence. Based on that, we infer that the directionality and the preferable regions of the sugar‐phosphate torsions, combined with the difference of purines from pyrimidines in ring shape, determines the sequence dependence of the structure of WC DNA. No such sequence dependence exists in dDMPs corresponding to other DNA conformations (e.g., Z‐family and Hoogsteen duplexes). Unlike other duplexes, WC helix is unique by its ability to match the local energy minima of the free single strand to the preferable conformations of the duplex. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 640–650, 2014.     

Effects of organic amines on α-aminoisobutyric acid uptake into the vacuole and on ethylene production by tomato pericarp slices

Experiments were conducted to test the possibility that organic amines inhibit ethylene production by inhibiting transport of the ethylene precursor, 1-aminocyclopro-pane-1-carboxylic acid (ACC), into the vacuole. α-Aminoisobutyric acid (αAIB) was used as a model substrate to study ACC uptake into the vacuole in relationship to ethylene production in pericarp slices of Lycopersicon esculentum Mill. cv. Liberty treated with and without organic amines and related substances. Organic amines (polyamines and other basic amines) inhibited αAIB uptake into the vacuole. These amines also enhanced ACC accumulation in the tissue and reduced the passive efflux of αAIB from the vacuole. Overall, ethylene production was inhibited. The inhibition of αAIB transport and of ethylene production followed a polyvalent cationic progression in the order polyamines > diamines> basic 1-amino acids. Ca²⁺, but not Mg²⁺, strongly stimulated αAIB uptake into the vacuole and ethylene production. At equal concentrations, Ca²⁺ counteracted the inhibitory effects of polyamines on both αAIB uptake and ethylene production. Competitive and irreversible inhibitors of polyamine biosynthesis stimulated αAIB uptake into the vacuole and ethylene production. The results indicate an apparent relationship between polyamines, ACC uptake into the vacuole and ethylene production.     

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 by DNA segment from a 5 portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs, and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.