Interaction of ecoP15I DNA methyltransferase with oligonucleotides containing the asymmetric sequence 5?-CAGCAG-3

EcoP15I DNA methyltransferase (Mtase) recognizes the asymmeteric sequence CAGCAG and catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the second adenine residue. We have investigated the DNA binding properties of EcoP15I DNA Mtase using gel mobility shift assays. EcoP15I DNA Mtase binds approximately threefold more tightly to DNA containing its recognition sequence, CAGCAG, than to non-specific sequences in the absence or presence of cofactors. Interestingly, in the presence of ATP the discrimination between specific and non-specific sequences increases significantly. These results suggest for the first time a role for ATP in DNA recognition by type III restriction-modification enzymes. In addition, we have shown that bromodeoxyuridine-containing oligonucleotides form complexes with EcoP15I DNA Mtase that are crosslinked upon irradiation. More importantly, we have shown that the crosslink site is at the site of DNA binding, since it can be suppressed by an excess of unmodified oligonucleotide. EcoP15I DNA Mtase exhibited Michaelis-Menten kinetics with both unmodified and bromodeoxyuridine-substituted DNA, with a higher specificity constant for the latter. Furthermore, gel mobility shift assays showed that proteolyzed EcoP15I DNA Mtase formed a specific complex with DNA, which had similar mobility as the native protein-DNA complex. Taken together these results form the basis fora detailed structure-function analysis of EcoP15I DNA Mtase.

Structure and stability of human telomeric sequence

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)(n). We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T(2)AG(3))(4), d(G(3)T(2)A)(3)G(3), and d(G(3)T(2)AG(3)) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G(3)T(2)A)(3)G(3) and d(T(2)AG(3))(4) assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G(3)T(2)AG(3)) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide D(G(3)T(2)AG(3)) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T(2)AG(3))(4) suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.

Lipid conformational nomenclature: a general method

We propose a conformational nomenclature for amphiphilic lipid molecules that is general and compatible with the stereospecific numbering scheme, in contrast to earlier methods in which discrepancies with the sn-scheme lead to contradictory assignments of the absolute configuration of the system. The present method can be rationally extended to different classes of lipids, both natural and synthetic. It is simple and provides a convenient framework for conformational studies on widely varying classes of lipids.

Molecular dynamics studies on nucleoside 2',3'-cyclic phosphates

2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP, 3'-UMP, A>p, G>p, U>p, C>p, GpA>p and UpA>p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.

Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

Helix termination and chain reversal: Crystal and molecular structure of the alpha,beta-dehydrooctapeptide Boc-Val Delta Phe-Phe-Ala-Leu-Ala-Delta Phe-Leu-OH

The crystal structure of the dehydro octapeptide Boc-Val-Delta Phe-Phe-Ala-Leu-Ala-Delta Phe-Leu-OH has been determined to atomic resolution by X-ray crystallographic methods. The crystals grown by slow evaporation of peptide solution in methanol/water are orthorhombic, space group P2(1)2(1)2(1). The unit cell parameters are a = 8.404(3), b = 25.598(2) and c = 27.946(3) Angstrom, Z = 4. The agreement factor is R = 7.58% for 3636 reflections having (\F-o\) greater than or equal to 3 sigma (\F-o\). The peptide molecule is characterised by a 3(10)-helix at the N-terminus and a pi-turn at the C-terminus. This conformation is exactly similar to the helix termination features observed in proteins. The pi-turn conformation observed in the octapeptide is in good agreement with the conformational features of pi-turns seen in some proteins. The alpha(L)-position in the pi-turn of the octapeptide is occupied by Delta Phe(7), which shows that even bulky residues can be accommodated in this position of the pi-turns. In proteins, it is generally seen that alpha(L)-position is occupied by glycine residue. No intermolecular head-to-tail hydrogen bonds are observed in solid state structure of the octapeptide. A water molecule located in the unit cell of the peptide molecule is mainly used to hold the peptide molecule together in the crystal. The conformation observed for the octapeptide might be useful to understand the helix termination and chain reversal in proteins and to construct helix terminators for denovo protein design.

Copper(I) complexes of modified nucleobases and vitamin B3 as potential chemotherapeutic agents: In vitro and in vivo studies

Three new complexes of Cu(I) have been synthesized using ancillary ligands like thiopyrimidine (tp) a modified nucleobase, and nicotinamide (nie) or vitamin B3, and characterized by spectroscopy and X-ray crystallography. In vitro cytotoxicity studies of the complexes on various human cancer cell lines such as Colo295, H226, HOP62, K562, MCF7 and T24 show that Cu(PPh3)(2)(tp)Cl] and Cu(PPh3)(2)(tp)ClO4 (2) have in vitro cytotoxicity comparable to cisplatin. Complex Cu(nic)(3)PPh3]ClO4 (3) is non-toxic and increases the life span by about 55 % in spontaneous breast tumor model. DNA binding and cleavage studies show that complex (3) binds to calf thymus DNA with an apparent binding constant of 5.9 x 10(5)M and completely cleaves super-coiled DNA at a concentration of 400 mu M, whereas complexes (1) and (2) do not bind DNA and do not show any cleavage even at 1200 mu M. Thus, complex (3) may exhibit cytotoxicity Via DNA cleavage whereas the mechanism of cytotoxicity of (1) and (2) probably involves a different pathway.

The structures of bacteriorhodopsin with different retinal-Schiff base orientations--computer modeling and energy minimization studies

Bacteriorhodopsin has been the subject of intense study in order to understand its photochemical function. The recent atomic model proposed by Henderson and coworkers based on electron cryo-microscopic studies has helped in understanding many of the structural and functional aspects of bacteriorhodopsin. However, the accuracy of the positions of the side chains is not very high since the model is based on low-resolution data. In this study, we have minimized the energy of this structure of bacteriorhodopsin and analyzed various types of interactions such as - intrahelical and interhelical hydrogen bonds and retinal environment. In order to understand the photochemical action, it is necessary to obtain information on the structures adopted at the intermediate states. In this direction, we have generated some intermediate structures taking into account certain experimental data, by computer modeling studies. Various isomers of retinal with 13-cis and/or 15-cis conformations and all possible staggered orientations of Lys-216 side chain were generated. The resultant structures were examined for the distance between Lys-216-schiff base nitrogen and the carboxylate oxygen atoms of Asp-96 - a residue which is known to reprotonate the schiff base at later stages of photocycle. Some of the structures were selected on the basis of suitable retinal orientation and the stability of these structures were tested by energy minimization studies. Further, the minimized structures are analyzed for the hydrogen bond interactions and retinal environment and the results are compared with those of the minimized rest state structure. The importance of functional groups in stabilizing the structure of bacteriorhodopsin and in participating dynamically during the photocycle have been discussed.

N-6-anisoyladenosine monohydrate

The title compound, C18H19N5O6. H2O, has a syn conformation about the glycosidic bond. Its furanose ring shows a C2'-endo-C3'-exo twist conformation and trans-gauche geometry about the C4'-C5' bond. The angle between the adenine base and the phenyl ring of the anisoyl group is 22.9 degrees. Adenine and anisoyl groups stack along the b axis at a separation of 3.4 Angstrom.

Design and structural analysis of hairpin-TFO for transcriptional activation of genes in S-cerevisiae

Triplex forming oligonucleotides (TFOs) have the potential to modulate gene expression. While most of the experiments are directed towards triplex mediated inhibition of gene expression the strategy potentially could be used for gene specific activation. In an attempt to design a strategy for gene specific activation in vivo applicable to a large number of genes we have designed a TFO based activator-target system which may be utilized in Saccharomyces cerevisiae or any other system where Gal4 protein is ectopically expressed. The total genome sequence of Saccharomyces cerevisiae and expression profiles were used to select the target genes with upstream poly (pu/py) sequences. We have utilized the paradigm of Gal4 protein and its binding site. We describe here the selection of target genes and design of hairpin-TFO including the targeting sequences containing polypurine stretch found in the upstream promoter regions of weakly expressed genes. We demonstrate, the formation of hairpin-TFO, its binding to Gal4 protein, its ability to form triplex with the target duplex in vitro, the effect of polyethylenimine on complex formation and discuss the implication on in vivo transcription activation.

DNA binding properties of novel dansylated distamycin analogues in which the fluorophore is directly conjugated to the N-methyl-pyrrole carboxamide backbone

Polyamides that are structural analogues of the naturally occurring DNA minor groove binding antibiotic distamycin (Dst) are promising candidates as gene modulators. Developing strategies for the large scale screening and monitoring of the cellular distribution of such ligands would aid the faster discovery of molecules, which would have eventual utility in molecular biology and medicine. Attachment of fluorescent tags would be a useful step towards this end. A fundamental question in this connection is whether the tag modifies the DNA binding affinity of the parent compounds. Towards answering this question, we have developed two oligopeptides that bear the dansyl (N, N-dimethylaminonaphthalene sulfonamido fluorophore) coupled directly to the N-terminus of the conjugated N-methylpyrrole carboxamide network, and possess three or four N-methyl pyrrole carboxamide units (abbreviated as Dn3 and Dn4 respectively). DNA binding abilities of these molecules were assessed from fluorescence titration experiments, duplex-DNA T-m analysis (employing both UV and fluorescence spectroscopy), induced circular dichroism measurements (ICD), salt dependence of ICD and apparent binding constant measurements (K-app) employing ethidium bromide (EtBr) displacement assay. Both these molecules 'reported' DNA binding in the form of an enhanced fluorescence emission. As judged from the ICD measurements, salt dependence of ICD, T-m analysis and K-app measurements, the binding affinities of the molecules that possessed dansyl group at their N-termini were lower than the ones with equivalent number of amide units, but possessed N-methylpyrrole carboxamide unit at their N-termini. These results would have implications in the future design of fluorescent polyamides.

Multicomponent solids of lamotrigine with some selected coformers and their characterization by thermoanalytical, spectroscopic and X-ray diffraction methods

The present study investigates the structural and pharmaceutical properties of different multicomponent crystalline forms of lamotrigine (LTG) with some pharmaceutically acceptable coformers viz. nicotinamide (1), acetamide (2), acetic acid (3), 4-hydroxy-benzoic acid (4) and saccharin (5). The structurally homogeneous phases were characterized in the solid state by DSC/TGA, FT-IR and XRD (powder and single crystal structure analysis) as well as in the solution phase. Forms 1 and 2 were found to be cocrystal hydrate and cocrystal, respectively, while in forms 3, 4 and 5, proton transfer was observed from coformer to drug. The enthalpy of formation of multicomponent crystals from their components was determined from the enthalpy of solution of the cocrystals and the components separately. Higher exothermic values of the enthalpy of formation for molecular complexes 3, 4 and 5 suggest these to be more stable than 1 and 2. The solubility was measured in water as well as in phosphate buffers of varying pH. The salt solvate 3 exhibited the highest solubility of the drug in water as well as in buffers over the pH range 7-3 while the cocrystal hydrate 1 showed the maximum solubility in a buffer of pH 2. A significant lowering of the dosage profile of LTG was observed for 1, 3 and 5 in the animal activity studies on mice.

Structure of tandem and its implication for bifunctional intercalation into dna

Quinoxaline antibiotics (Fig. 1a, b) form a useful group of compounds for the study of drug–nucleic acid interactions1,2. They consist of a cross-bridged cyclic octadepsipeptide, variously modified, bearing two quinoxaline chromophores. These antibiotics intercalate bifunctionally into DNA2,3 probably via the narrow groove, forming a complex in which, most probably, two base pairs are sandwiched between the chromophores4,5. Depending on the nature of their sulphur-containing cross-bridge and modifications to their amino acid side chains, they display characteristic patterns of nucleotide sequence selectivity when binding to DNAs of different base composition and to synthetic polydeoxynucleotides4,6,7. This specificity has been tentatively ascribed to specific hydrogen-bonding interactions between functional groups in the DNA and complementary moieties on the peptide ring2,4,5. Variations in selectivity have been attributed both to changes in the conformation of the peptide backbone6 and no modifications of the cross-bridge7. These suggestions were made, however, in the absence of firm knowledge about the three-dimensional structure and conformation of the antibiotic molecules. We now report the X-ray structure analysis of the synthetic analogue of the antibiotic triostin A, TANDEM (des-N-tetramethyl triostin A) (Fig. 1c), which binds preferentially to alternating adenine-thymine sequences7. The X-ray structure provides a starting point for exploring the origin of this specificity and suggests possible models for the binding of other members of the quinoxaline series.

Structure of DNA-functionalized dendrimer nanoparticles

Atomistic molecular dynamics simulations have been carried out to reveal the characteristic features of ethylenediamine (EDA) cored protonated (corresponding to neutral pH) poly amido amine (PAMAM) dendrimers of generation 3 (G3) and 4 (G4) that are functionalized with single strand DNAs (ssDNAs). The four ssDNA strands that are attached via an alkythiolate [-S(CH(2))(6)-] linker molecule to the free amine groups on the surface of the PAMAM dendrimers are observed to undergo a rapid conformational change during the 25 ns long simulation period. From the RMSD values of ssDNAs, we find relative stability in the case of purine rich (having more adenine and guanine) ssDNA strands than pyrimidine rich (thymine and cytosine) ssDNA strands. The degree of wrapping of ssDNA strands on the dendrimer molecule was found to be influenced by the charge ratio of DNA and the dendrimer. As the G4 dendrimer contains relatively more positive charge than G3 dendrimer, we observe extensive wrapping of ssDNAs on the G4 dendrimer than G3 dendrimer. This might indicate that DNA functionalized G3 dendrimer is more suitable to construct higher order nanostructures. The linker molecule was also found to undergo drastic conformational change during the simulation. During nanosecond long simulation some portion of the linker molecule was found to be lying nearly flat on the surface of the dendrimer molecule. The ssDNA strands along with the linkers are seen to penetrate the surface of the dendrimer molecule and approach closer to the center of the dendrimer indicating the soft sphere nature of the dendrimer molecule. The effective radius of DNA-functionalized dendrimer nanoparticles was found to be independent of base composition of ssDNAs and was observed to be around 19.5 angstrom and 22.4 angstrom when we used G3 and G4 PAMAM dendrimers as the core of the nanoparticle respectively. The observed effective radius of DNA-functionalized dendrimer molecules apparently indicates the significant shrinkage in the structure that has taken place in dendrimer, linker and DNA strands. As a whole our results describe the characteristic features of DNA-functionalized dendrimer nanoparticles and can be used as strong inputs to design effectively the DNA-dendrimer nanoparticle self-assembly for their active biological applications.