Comparative study of the interaction of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP) in its free base and Fe derivative form with oligo(dA.dT)15 and oligo(dG.dC)15

Interaction between a cationic porphyrin and its ferric derivative with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ was studied by UV–vis spectroscopy, resonance light scattering (RLS), and circular dichroism (CD) at different ionic strengths; molecular docking and molecular dynamics simulation were also used for completion. Followings are the observed changes in the spectral properties of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP), as a free-base porphyrin with no axial ligand, and its Fe derivative (FeTMAP) upon interaction with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅: (1) the substantial red shift and hypochromicity at the Soret maximum in the UV–vis spectra; (2) the increased RLS intensity by increasing the ionic strength; and (3) an intense bisignate excitonic CD signal. All of them are the reasons for TMAP and FeTMAP binding to oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ with the outside binding mode, accompanied by the self-stacking of the ligands along the oligonucleotide helix. The CD results demonstrated a drastic change from excitonic in monomeric behavior at higher ionic strengths, which indicates the groove binding of the ligands with oligonucleotides. Molecular docking also confirmed the groove binding mode of the ligands and estimated the binding constants and energies of the interactions. Their interaction trend was further confirmed by molecular dynamics technique and structure parameters obtained from simulation. It showed that TMAP reduced the number of intermolecular hydrogen bonds and increased the solvent accessible surface area in the oligonucleotide. The self-aggregation of ligands at lower concentrations was also confirmed.     

Chain length characterization of oligodeoxyribonucleotides by analytical ultracentrifugation

Analytical ultracentrifugation was used to determine chain length of oligomers in the deoxyribonucleotide series. Uncertainties with respect to partial specific volume of the solute and questions of charge compel a semiempirical approach: variations of these parameters introduce fluctuations well within the experimental error of the method. Measurements of the sedimentation equilibria for three homooligomer series—d(pT)_n, d(pA)_n and d(pC)_n—as well as a number of oligomers of varying base composition generated straight-line plots passing through the origin.     

Catalytic antibodies from HIV-infected patients specifically hydrolyzing viral integrase suppress the enzyme catalytic activities

Human immunodeficiency virus type 1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. It was shown previously that IN preincubation with various oligodeoxynucleotides (ODNs) induces formation of dimers and oligomers of different gyration radii; only specific ODNs stimulate the formation of catalytically active dimers. Here we have shown that preincubation of IN with specific and nonspecific ODNs leads to a significant and comparable decrease in its hydrolysis by chymotrypsin, while nonspecific ODNs protect the enzyme from the hydrolysis by trypsin worse than specific ODNs; all ODNs had little effect on the IN hydrolysis by proteinase K. In contrast to canonical proteweases, IgGs from HIV‐infected patients specifically hydrolyze only IN. While d(pT)_n markedly decreased the IgG‐dependent hydrolysis of IN, d(pA)_n and d(pA)_n?d(pT)_n demonstrated no detectable protective effect. The best protection from the hydrolysis by IgGs was observed for specific single‐ and especially double‐stranded ODNs. Although IN was considerably protected by specific ODNs, proteolytic IgGs and IgMs significantly suppressed both 3′‐processing and integration reaction catalyzed by IN. Since anti‐IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. Copyright © 2011 John Wiley & Sons, Ltd.     

Hybridization of Oligo(dT) to RNA on nitrocellulose

Quantitation of mRNA immobilized on nitrocellulose filters is an essential aspect of some studies in molecular biology. Hybridization of oligo(dT)₁₈ to the poly(A) tails of mRNA can be used to measure filter-bound mRNA and thus provides a basis for comparing abundance of specific mRNAs. Hybridization rate of ³²P-labeled oligo(dT)₁₈ in 0.75 M NaCl, 75 mM sodium citrate, pH 7 (5 × SSC) to immobilized RNA was maximal at 25°C. Filters were fully hybridized under these conditions within 1 hr when the oligo(dT)₁₈ concentration was 10 pmol/ml or higher. Salt dependence of the dissociation temperature (T_d) of oligo(dT)₁₈:RNA duplex on filters was described by the equation T_d = 42 − 20log₁₀[molar Na⁺] (°C). With stringent washing of the duplex (four 5-min washes in 2 × SSC at room temperature), oligo(dT)₁₈ gave no signal with plasmid DNA, rRNA, or tRNA. We have found that olig(dT)₁₈ can be used to normalize signal strengths rapidly and conveniently from total or oligo(dT)-selected eukaryotic RNA.     

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein

Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔH_obs, and accounts for as much as a half of the observed heat capacity change, ΔC_p. However, there is still a substantial ΔC_p associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)₃₄ to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)₃₄ binding over this entire pH range, with contributions from at least three sets of protonation sites (pK_a1 = 5.9–6.6, pK_a2 = 8.2–8.4, and pK_a3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔC_p for the SSB‐dT(pT)₃₄ interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol⁻¹ K⁻¹) accounts for as much as half of the total ΔC_p. The values of the “intrinsic” ΔC_p,0 range from −210 ± 33 cal mol⁻¹ °K⁻¹ to −237 ± 36 cal mol⁻¹K⁻¹, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔC_p, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc.     

Proton magnetic resonance study of nucleosides,nucleotides, and dideoxynucleoside monophosphates containing a syn pyrimidine base

Proton magnetic resonance data have been obtained for 6-methyl-2′-deoxyuridine (dT*), its 3′- and 5′-monophosphates, and its 3′,5′-diphosphate, as well as for the corresponding thymine derivatives. The synthesis of the dideoxynucleoside monophosphates—d(TpT), d(T*pT), d(TpT*), and d(T*pT*)—was accomplished, and spectral data were obtained for these four dimers. The data show that the 6-methyluracil base prefers the syn conformation about the N-glycosyl bond at the monomer and dimer levels. The presence of the syn base leads to increases in the cis couplings of the sugar ring, J_1′2″ and J_2′3′, which indicate a trend towards eclipsing of the substituents on the C1′-C2′ and C2′-C3′ fragments. This trend is discussed in terms of changes in the pseudorotational parameters which describe the pucker of the ring. The syn base destabilizes the g⁺ conformer about the C4′-C5′ bond, leading to a preference for the t conformer in all dT* residues at the monomer and dimer levels. Preliminary work on the formation of cyclobutane-type photodimers in d(T*pT) and d(T*pT*) is discussed and presented as evidence for the capability of the syn 6-methyluracil base to form base-stacked complexes.     

One- and two-dimensional NMR studies on the conformation of DNA containing the oligo(dA)oligo(dT) tract.

The resonances of the imino protons and all of the non-exchangeable protons (except for H5'/H5') of d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) have been assigned by means of one- and two-dimensional NMR spectroscopies. Qualitative analyses showed that the overall structure is of the B-form, but local conformational deviations exist. The NOEs between the imino protons of thymines and H2 of adenines suggest that the A-T base pairs are propeller-twisted to almost the same degree as in crystals. A remarkable chemical shift of H1' was observed for the residue located just before the oligo(dA)oligo(dT) tract, suggesting the presence of conformational discontinuity at the junctions between the oligo(dA)oligo(dT) tract and the other portions. Analyses of cross peaks in NOESY spectra between H2 of adenines and H1' of the 3'-neighbouring residues on the complementary strand revealed that the minor groove of the oligo(dA)oligo(dT) tract is narrow and compressed gradually, from 5' to 3', along the tract.     

Interaction of single-stranded DNA with the second DNA-binding site of the RecA nucleoprotein filament

RecA first forms a filament on single-stranded DNA (ssDNA), thereby forming the first site for ssDNA binding and, simultaneously, the second site for binding double-stranded DNA (dsDNA). Then, the nucleoprotein filament interacts with dsDNA, although it can bind ssDNA as well. The resulting complex searches for homology sites and performs strand exchange between homologous DNA molecules. The interaction of various ssDNAs with the second DNA-recognizing site of RecA was studied by gradually increasing the structural complexity of the DNA ligand. Recognizing ssDNA with the second site, the protein interacts with each nucleotide of the ligand, forming contacts with both internucleotide phosphate groups and nitrogen bases. Pyrimidine oligonucleotides d(pC) _n and d(pT) _n interacted with the second site of the RecA filament more efficiently than d(pA) _n did. This was due to a more efficient interaction of the RecA filament with the 5′-terminal nucleotide of pyrimidinic DNA and to the difference in specific conformational changes of the nucleoprotein filament in the presence of purinic and pyrimidinic DNAs. A comparison of thermodynamic characteristics of DNA recognition at the first and second DNA-binding sites of the filament showed that, at n > 10, d(pC) _n and d(pN) _n were bound at the second site less tightly than at the first site. At n > 20, the second site bound d(pA) _n more efficiently than the first site. The difference in d(pN) _n affinity for the first and second sites increased monotonically with increasing n. Possible mechanisms of a RecA-dependent search for homology and DNA strand exchange are discussed.     

Adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates.

Isothermal titration calorimetry (ITC) was used to test the hypothesis that the relatively small enthalpy change (DeltaHobs) and large negative heat capacity change (DeltaCp,obs) observed for the binding of the Escherichia coli SSB protein to single-stranded (ss) oligodeoxyadenylates result from the temperature-dependent adenine base unstacking equilibrium that is thermodynamically coupled to binding. We have determined DeltaH1,obs for the binding of 1 mole of each of dT(pT)34, dC(pC)34, and dA(pA)34 to the SSB tetramer (20 mM NaCl at pH 8.1). For dT(pT)34 and dC(pC)34, we found large, negative values for DeltaH1,obs of -75 +/- 1 and -85 +/- 2 kcal/mol at 25 degrees C, with DeltaCp,obs values of -540 +/- 20 and -570 +/- 30 cal mol-1 K-1 (7-50 degrees C), respectively. However, for SSB-dA(pA)34 binding, DeltaH1,obs is considerably less negative (-14 +/- 1 kcal/mol at 25 degrees C), even becoming positive at temperatures below 13 degrees C, and DeltaCp,obs is nearly twice as large in magnitude (-1180 +/- 40 cal mol-1 K-1). These very different thermodynamic properties for SSB-dA(pA)34 binding appear to result from the fact that the bases in dA(pA)34 are more stacked at any temperature than are the bases in dC(pC)34 or dT(pT)34 and that the bases become unstacked within the SSB-ssDNA complexes. Therefore, the DeltaCp,obs for SSB-ssDNA binding has multiple contributions, a major one being the coupling to binding of a temperature-dependent conformational change in the ssDNA, although SSB binding to unstacked ssDNA still has an "intrinsic" negative DeltaCp,0. In general, such temperature-dependent changes in the conformational "end states" of interacting macromolecules can contribute significantly to both DeltaCp,obs and DeltaHobs.     

Effective solvent systems for separation and an improved detection method for amino acids by paper chromatoghraphy

Binding of poly(A)-containing RNP to oligo(dT)-cellulose has been investigated as a function of mono- and divalent ion concentration. 80–90% binding was obtained either in high (500 mM) or in moderate NaCl concentrations in the presence of 5 mM MgCl₂. At 40 mM NaCl and 5 mM MgCl₂ poly(A)⁺-RNP exhibit approximately t he same stability as poly(A)⁺-RNA in binding to oligo(dT)-cellulose with a melting temperature of 41 and 45°C, respectively, indicating that the protein moeity has no effect on the ribonucleoprotein binding in these conditions. Differences were observed int he elution of poly(A)⁺-RNA and poly(A)⁺-RNP from oligo(dT)-cellulose in buffer without salts. Poly(A)⁺-RNA was completely removed at 4°C whereas the melting temperature of poly(A)⁺-RNP was only decreased to 34°C. The isolation of poly(A)⁺-RNP by thermal elution from oligo(dT)-cellulose is described.     

Oligodeoxynucleotide base recognition by steroid hormone receptors

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E₂R) the relative binding order, in prior studies, was oligo(dG) > oligo(dT) ≧ oligo(dC) > > oligo(dA) > oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1–0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E₂R to chaotropic salts such as SCN^?, ClO₄^? and NO₃^? as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E₂R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37°C was not altered by addition of H2B. Treatment of uterine E₂R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.     

Circular dichroism studies of α-aminoisobutyric acid-containing peptides: Chain length and solvent effects in alternating Aib-L-Ala and Aib-L-Val sequences

The CD spectra of the peptides Boc-X-(Aib-X)_n-OMe (n = 1, 2, 3) and Boc-(Aib-X)₅-OMe, where X = L -Ala or L -Val have been examined in several solvents. The X = Ala and Val peptides behave similarly in all solvents, suggesting that the Aib residues dominate the folding preferences of these peptides. The decapeptides adopt helical conformations in methanol and trifluoroethanol, with characteristic negative CD bands at 222 and 205 nm. In the heptapeptides, similar spectra with reduced intensities are observed. Comparison with nmr studies suggest that estimates of helical content in oligopeptides by CD methods may lead to erroneous conclusions. The pentapeptides yield solvent-dependent spectra indicative of conformational perturbations. Peptide association in dioxane results in an unusual spectrum with a single negative band at 210 nm for the decapeptides. Disaggregation is induced by the addition of methanol or water to dioxane solutions. Aggregation of the heptapeptides is less pronounced in dioxane, suggesting that a critical helix length may be necessary to promote association stabilized by helix dipole–dipole interactions.     

Optical activity of single-stranded polydeoxyadenylic and polyriboadenylic acids; dependence of adenine chromophore cotton effects on polymer conformation

Circular dichroism (CD) curves are reported for poly dA, (pdA)₆, (pdA)₂, poly A, ApAp, ApA, AMP, dApA, pdApA, A-2′-O-methyl pA, and A-2′-O-methyl pAp. Analysis of these curves indicated the presence of single CD bands at 228–230 mμ and at 278–280 mμ in oligomers longer than dinucleotides. In the case of dinucleotides and mononucleotides (from the literature, in addition to those studied here), the 230 mμ CD of band appears but the 280 mμ CD band does not. We assign the 230 mμ band to a very weak π–π* transition at this wavelength. From theoretical considerations, we show that the 280 mμ band is not an exciton component of the strong π–π* transition at 260 mμ in adenine. We conclude that the 280 mμ CD band must be assigned to a distinct absorption, not previously reported, which we suggest arises from an n–π* transition. The fact that the n–π* CD band at 280 mμ is not seen in mononucleotides or dinucleotides is ascribed to solvation of the adenine ring by water, which shifts the band to shorter wavelengths. Therefore, only interior residues of oligomers have the 280 mμ band, and the optical activity of a polymer cannot be computed from that of a dinucleotide, by using a nearest-neighbor approximation. The existence of this end effect hag been tested, by taking it into account in computing the rotational strengths of the 278 mμ n–π* transition for several oligomers; it is pointed out that a more sensitive test of this end effect would require CD data for the oligo dA series of 3 to 5 residues. We speculate about the structural and optical differences between poly dA and poly A, and point out the need for a theoretical treatment of n–π* Cotton effects in polynucleotides.     

Gene 5 protein of bacteriophage fd: A dimer which interacts Co-operatively with DNA

Isolated gene 5 protein from bacteriophage fd-infected Escherichia coli has been shown by sedimentation equilibrium to exist primarily as a dimer under non-denaturing conditions. The dimer was stable under conditions of high ionic strength, extremes in pH, dilution to 0.075 mg/ml, and increased temperature. Gene 5 protein did not undergo the indefinite self-association observed with gene 32 protein.Three lines of evidence for co-operative binding of gene 5 protein to DNA were developed. First, the interaction between gene 5 protein and phage T4 DNA was examined using a nitrocellulose filter assay. Scatchard plots of the binding data indicated that the interaction was co-operative. Similar results were obtained with gene 32 protein. Second, the co-operative binding of both proteins to DNA was shown by the sensitivity of the protein-DNA interaction to increasing ionic strength at various ratios of protein to DNA. Finally, by using the cross-linking agent, dimethyl suberixmidate, oligomeric structures containing at least seven monomers were found when the DNA was less than saturated.The possibility that gene 5 protein dimers undergo indefinite self-association in the presence of oligonucleotides was examined by sedimentation equilibrium. With oligo[d(pT)₄], the protein dimer was complexed with this oligonucleotide but no self-association was observed. With oligo[d(pT)₈], gene 5 protein formed tetramers, but no significant indefinite association was noted. These results do not suggest a DNA-induced conformational change, which results in indefinite association. A model for the co-operative binding of gene 5 protein to DNA is presented.     

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K _m [ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.     

Substrate specificity of Ca2+,Mg2+-dependent DNAase from sea urchin (Strongylocentrotus intermedius) embryos

Ca2+,Mg2+-dependent DNAse from sea urchin embryos is specific to the secondary structure of substrates irrespective of the nature of activating cations. The enzyme does not split synthetic single-stranded oligo and polynucleotides, such as d(pTpTpTpCpC), d(pGpGpTpTpT). d(pApApTpTpC), d(pGpApApTpTpC), d(pA)5-poly(dT), d(pApApTpTpC)-poly(dT), poly(dA) and poly (dT) and hydrolyses the double-stranded substrates poly d(AT), poly (dA) . poly (dT) and highly polymerized DNA. Native double-stranded DNA from salmon and phage T7 is split by the enzyme at a higher rate than that of denaturated DNA of salmon and single-stranded DNA of phage M13. The high rate of poly(dA) . poly(dT) and poly d(AT) hydrolysis and the stability of poly(dG) . poly(dC) to the effect of the enzyme suggest a certain specificity of the enzyme to the nature of nitrogenous bases at the hydrolyzed phosphodiester bond of the substrate.     

Comparison of initiating abilities of primers of different length in polymerization reactions catalyzed by DNA polymerases from thermoacidophilic archaebacteria

Optimal conditions for polymerization reaction catalyzed on poly(dA) and poly(dT) templates by DNA polymerases from thermoacidophilic archaebacteria--DNA polymerase A from Sulfolobus acidocaldarius and DNA polymerase B from Thermoplasma acidophilum--have been established. Values of Km and Vmax (60 degrees C) for a set of primers d(pA)n and d(pT)n have been estimated. Minimal primers for both enzymes are dNMP. Lengthening of primers by each mononucleotide increases their affinity about 2.16-fold. Linear dependence of log Km and of log vmax on the number of mononucleotide links in primers (n) has breaking point at n = 10. The value of Vmax is about 20% of that for decanucleotide. The affinity of the primer d(pA)9p(rib*) with a deoxyribosylurea residue at the 3'-end does not differ essentially from that of d(pA)9. Substitution of the 3'-terminal nucleotide of a complementary primer for a noncomplementary nucleotide, e.g., substitution of 3'-terminal A for C in d(pA)10 in the reaction catalyzed on poly(dT), decreases the affinity of a primer by one order of magnitude.     

A novel intramolecular G-quartet-containing fold of single-stranded d(GT)8 and d(GT)16 oligonucleotides

Human genome is shown to be enriched with (GT)_n stretches of lengths from 8 to 20 dinucleotides. Low temperature (T ≤ 10 °C) conformations of d(GT)_n oligonucleotides (n = 7, 8, 12, 16, 20) were studied by means of circular dichroism (CD), thermal melting, ethidium bromide (EtBr) probing and single nucleotide substitutions. Rotational relaxation times for EtBr:d(GT)_n complexes confirmed a monomolecular state of the oligonucleotides. CD spectra indicated involvement of all guanines of d(GT)₈ and d(GT)₁₆ in G-quartets, while dT(GT)₇, d(GT)₁₂ and d(GT)₂₀ were shown to be only partially ordered. The schemes of the d(GT)₈ and d(GT)₁₆ folds are suggested.     

Binding geometries of triple helix selective benzopyrido [4,3-b]indole ligands complexed with double- and triple-helical polynucleotides

The binding modes of three benzopyrido [4,3-b]indole derivatives (and one benzo[-f]pyrido [4-3b] quinoxaline derivative) with respect to double helical poly(dA) · poly(dT) and poly[d(A-T)]₂ and triple-helical poly(dA) · 2poly(dT) have been investigated using linear dichroism (LD) and CD: (I) 3-methoxy-11-amino-BePI where BePI = (7H-8-methyl-benzo[e]pyrido [4,3-b]indole), (II) 3-methoxy-11-[(3′-amino) propylamino]-BePI, (III) 3-methoxy-7-[(3′-diethylamino)propylamino] BgPI where BgPI = (benzo[g]pyrido[4,3-b]indole), and (IV) 3-methoxy-11-[(3′-amino)propylamino] B f P Q where B f P Q = {benzo[-f]pyrido[4-3b]quinoxaline}. The magnitudes of the reduced LD of the electronic transitions of the polynucleotide bases and of the bound ligands are generally very similar, suggesting an orientation of the plane of the ligands' fused-ring systems preferentially perpendicular to the helix axis. The LD results suggest that all of the ligands are intercalated for all three polynucleotides. The induced CD spectrum of the BePI chromophore in the (II-BePI)-poly[d(A-T)]₂ complex is almost a mirror image of that for the (I-BePI)-poly(dA) · poly(dT) and (I-BePI)-poly(dA) · 2poly(dT) complexes, suggesting an antisymmetric orientation of the BePI moiety upon intercalation in poly[d(A-T)]₂ compared to the other polynucleotides. The induced CD of I-BePI bound to poly(dA) · 2poly(dT) suggests a geometry that is intermediate between that of its other two complexes. The concluded intercalative binding as well as the conformational variations between the different BePI complexes are of interest in relation to the fact that BePI derivatives are triplex stabilizers. © 1997 John Wiley & Sons, Inc. Biopoly 42: 101–111, 1997     

Effect of bases noncomplementary to the template on the effectiveness of primer interaction with DNA-polymerase alpha from the human placenta

The comparison of the Km and Vmax values for the various primers was carried out. The primers were either completely complementary to the template or contained the non-complementary bases in different positions from the 3'-end. The number of the bases from the 3'-end to the noncomplementary nucleotide but not the primers length was supposed to determine the efficiency of the interaction of the primers containing noncomplementary bases with the enzyme. The Km values for d[(pC) (pT)7] (1.2 microM), d[(pC)3(pT)7] (2.5 microM, d[(pT)2pC(pT)7] (1.4 microM)d[(pT)4pC(pT)5(4.3 microM); d[(pT)7pC(pT)2] (11 microM) are comparable with the Km values for d(pT)7 (1.4 microM); d(pT)5 (4.2 microM) and d(pT)3 (15 mkM), respectively, but not for the decathymidilate d[(Tp)9T] (0.23 microM). The complementary interaction between the first nucleotide from the 3'-end of the primer and the template appear to play the particular role in the interaction of the enzyme with the primer. The Km values for d[(pT)10pC] and d[(pA)9pC] (with the corresponding templates) are 38 and 6 times the ones for d[(Tp)10T] and d(pA)10. However, the Km values for d[(pA)9p(rib)] (0.56 microM) which contains the deoxyribozylurea residue at the 3'-end is practically equal to the Km for d(pA)9 (0.56 microM). The Vmax values for d[(pT)10pC] and d[(pA)9pC] are 1.7 and 2.3 times the values for d[(Tp)10T] and d(pA)10, respectively. The primer affinity decreases, just as its conversion rate increases when the noncomplementary base in the primer is transferred from the 5'-to 3'-end; that results in the rate of primers elongation decrease in total.