Coralyne binds tightly to both T.A.T- and C.G.C(+)-containing DNA triplexes

Coralyne is a DNA-binding antitumor antibiotic whose structure contains four fused aromatic rings. The interaction of coralyne with the DNA triplexes poly(dT).poly(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[d(C+T)] was investigated by using three techniques. First, Tm values were measured by thermal denaturation analysis. Upon binding coralyne, both triplexes showed Tm values that were increased more than those of the corresponding duplexes. A related drug, berberinium, in which one of the aromatic rings is partially saturated, gave much smaller changes in Tm. Second, the fluorescence of coralyne is quenched in the presence of DNA, allowing the measurement of binding parameters by Scatchard analysis. The binding isotherms were biphasic, which was interpreted in terms of strong intercalative binding and much weaker stacking interactions. In the presence of 2 mM Mg2+, the binding constants to poly(dT).poly-(dA).poly(dT) and poly[d(TC)].poly[d(GA)].poly[(C+T)] were 3.5 x 10(6) M-1 and 1.5 x 10(6) M-1, respectively, while the affinity to the parent duplexes was at least 2 orders of magnitude lower. In the absence of 2 mM Mg2+, the binding constants to poly[d(TC)].poly[d(GA)].poly[d(C+T)] and poly-[d(TC)].poly[d(GA)] were 40 x 10(6) M-1 and 15 x 10(6) M-1, respectively. Thus coralyne shows considerable preference for the triplex structure but little sequence specificity, unlike ethidium, which will only bind to poly(dT).poly(dA).poly(dT). Further evidence for intercalation of coralyne was provided by an increase in the relative fluorescence quantum yield at 260 nm upon binding of coralyne to triplexes as well as an absence of quenching of fluorescence in the presence of Fe[(CN)6]4-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of the antiviral quinoxaline derivative 9-OH-B220 [2, 3-dimethyl-6-(dimethylaminoethyl)- 9-hydroxy-6H-indolo-[2, 3-b]quinoxaline] with duplex and triplex forms of synthetic DNA and RNA

The binding of an antiviral quinoxaline derivative, 2,3-dimethyl- 6 - (dimethylaminoethyl) - 9 - hydroxy - 6H - indolo - [2,3 - b]quinoxaline (9-OH-B220), to synthetic double and triple helical DNA (poly(dA).poly(dT) and poly(dA).2poly(dT)) and RNA (poly(rA). poly(rU) and poly (rA).2poly(rU)) has been characterized using flow linear dichroism (LD), circular dichroism (CD), fluorescence spectroscopy, and thermal denaturation. When either of the DNA structures or the RNA duplex serve as host polymers a strongly negative LD is displayed, consistent with intercalation of the chromophoric ring system between the base-pairs/triplets of the nucleic acid structures. Evidence for this geometry also includes weak induced CD signals and strong increments of the fluorescence emission intensities upon binding of the drug to each of these polymer structures. In agreement with intercalative binding, 9-OH-B220 is found to effectively enhance the thermal stability of both the double and triple helical states of DNA as well as the RNA duplex. In the case of poly(dA).2poly(dT), the drug provides an unusually large stabilization of its triple helical state; upon binding of 9-OH-B220 the triplex-to-duplex equilibrium is shifted towards higher temperature by 52.5 deg. C in a 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. When triplex RNA serves as host structure, LD indicates that the average orientation angle between the drug chromophore plane and the helix axis of the triple helical RNA is only about 60 to 65 degrees. Moreover, the thermal stabilizing capability, as well as the fluorescence increment, CD inducing power and perturbations of the absorption envelope, of 9-OH-B220 in complex with the RNA triplex are all less pronounced than those observed for the complexes with DNA and duplex RNA. These features indicate binding of 9-OH-B220 in the wide and shallow minor groove of poly(rA).2poly(rU). Based on the present results, some implications for the applications of this low-toxic, antiviral and easily administered drug in an antigene strategy, as well as its potential use as an antiretroviral agent, are discussed.     

Different effects of nonintercalative antitumor drugs on DNA triple helix stability: SN-18071 promotes triple helix formation

The interaction of the nonintercalating bisquaternary ammonium heterocyclic drugs SN-18071 and SN-6999 with a DNA triple helix has been studied using thermal denaturation and CD spectroscopy. Our data show, that both minor groove binders can bind to the triple helix of poly(dA).2poly(dT) under comparable ionic conditions, but they influence the stability of the triplex relative to the duplex structure of poly(dA).poly(dT) in a different manner. SN-18071, a ligand devoid of forming hydrogen bonds, can promote triplex formation and thermally stabilizes it up to 500 mM Na+ concentration. SN-6999 destabilizes the triplex to duplex equibilirium whereas it stabilizes the duplex. The binding constant of SN-18071 is found to be greater than that to the duplex. The stabilizing effect of SN-18071 is explained by electrostatic interactions of three ligand molecules with the three grooves of the triple stranded structure. From the experiments it is concluded that SN-6999 binds to the triplex minor groove thereby destabilizing the triplex similar as previously reported for netropsin.     

Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.     

Sequence-selective biosensor for DNA based on electroactive hybridization indicators

Deoxyribonucleic acid was covalently immobilized onto oxidized glassy carbon electrode surfaces that had been activated using 1-[3-(dimethylamino)-propyl]-3-ethylcarbodimide hydrochloride and N-hydroxysulfosuccinimide. This reaction is selective for immobilization through deoxyguanosine (dG) residues. Immobilized DNA was detected voltammetrically, using tris (2,2'-bipyridyl)cobalt(III) perchlorate and tris (1,10-phenanthroline)cobalt(III) perchlorate (Co(bpy)3(3+) and Co(phen)3(3+). These complexes are reversibly electroactive (1e-) and preconcentrate at the electrode surface through association with double-stranded DNA. Voltammetric peak currents obtained with a poly(dG)poly(dC)-modified electrode depend on [Co(bpy)3(3+)] and [Co(phen)3(3+)] in a nonlinear fashion and indicate saturation binding with immobilized DNA. Voltammetric peak currents for Co(phen)3(3+) reduction were used to estimate the (constant) local DNA concentration at the modified electrode surface; a binding site size of 5 base pairs and an association constant of 1.74 x 10(3) M(-1) yield 8.6 +/- 0.2 mM base pairs. Cyclic voltammetric peak separations indicate that heterogeneous electron transfer is slower at DNA-modified electrodes than at unmodified glassy carbon electrodes. A prototype sequence-selective DNA sensor was constructed by immobilizing a 20-mer oligo (deoxythymidylic acid) (oligo(dT)20), following its enzymatic elongation with dG residues, which yielded the species oligo(dT)20(dG)98. Cyclic voltammograms of 0.12 mM Co(bpy)3(3+) obtained before and after hybridization with poly-(dA) and oligo(dA)20 show increased cathodic peaks after hybridization. The single-stranded form is regenerated on the electrode surface by rinsing with hot deionized water. These results demonstrate the use of electroactive hybridization indicators in a reusable sequence-selective biosensor for DNA.     

Effects of hydration, ion release, and excluded volume on the melting of triplex and duplex DNA

The stability of DNA duplex and triplex structures not only depends on molecular forces such as base pairing or tripling or electrostatic interactions but also is sensitive to its aqueous environment. This paper presents data on the melting of Escherichia coli and poly(dA).poly(dT) duplex DNA and on the poly(dT).poly(dA). poly(dT) triplex in a variety of media to assess the contributions from the osmotic status and salt content of the media. The effects of volume exclusion on the stability of the DNA structures are also studied. From thermal transition measurements in the presence of low-molecular weight osmotic stressors, the number of water molecules released upon melting is found to be four waters per base pair for duplex melting and one water for the conversion of triplex to single-strand and duplex. The effects of Na+ counterion binding are also determined in ethylene glycol solutions so that the variation of counterion binding with water activity is evaluated. The data show that there is a modest decrease in the extent of counterion binding for both duplex and triplex as water activity decreases. Finally, using larger polyethylene glycol cosolutes, the effects on melting of volume exclusion by the solutes are assessed, and the results correlated with simple geometric models for the excluded volume. These results point out that DNA stability is sensitive to important conditions in the environment of the duplex or triplex, and thus, conformation and reactivity can be influenced by these solution conditions.     

Orthodontic treatment and temporomandibular disorders

Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)2, poly(dA).poly(dT), poly(dG.dC)2 and poly(dG).poly(dC). These spectra are interpreted as evidence of multiple outside binding modes with poly(dA).poly(dT), and of evidence for an outside binding mode with Poly(dG.dC). Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.     

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-t rimethylammoniumethyl-amine)phenyl]porphy rin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine )phenyl]porphyrin (TthetaF4TAP) has been synthesized. The binding interactions of TthetaF4TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TthetaF4TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TthetaF4TAP binds to calf thymus DNA, [poly(dA-dT)]2, and [poly(dG-dC)]2 in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TthetaF4TAP/CT DNA resembled that of TthetaF4TAP/[poly(dA-dT)]2, suggesting that TthetaF4TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TthetaF4TAP binds preferentially to [poly(dA-dT)]2 over [poly (dG-dC)]2. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TthetaF4TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TthetaF4TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation.     

DNA minor groove binding-directed poisoning of human DNA topoisomerase I by terbenzimidazoles

We have employed a broad range of spectroscopic, calorimetric, DNA cleavage, and DNA winding/unwinding measurements to characterize the DNA binding and topoisomerase I (TOP1) poisoning properties of three terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB), terbenzimidazole (TB), and 5-(naphthyl[2,3-d]imidazo-2-yl)bibenzimidazole (5NIBB), which differ with respect to the substitutions at their C5 and/or C6 positions. Our results reveal the following significant features. (i) The overall extent to which the three terbenzimidazole analogues poison human TOP1 follows the hierarchy 5PTB > TB > 5NIBB. (ii) The impact of the three terbenzimidazole analogues on the superhelical state of plasmid DNA depends on the [total ligand] to [base pair] ratio (rbp), having no effect on DNA superhelicity at rbp ratios < or = 0.1, while weakly unwinding DNA at rbp ratios > 0.1. This weak DNA unwinding activity exhibited by the three terbenzimidazoles does not appear to be correlated with the abilities of these compounds to poison TOP1. (iii) Upon complexation with both poly(dA).poly(dT) and salmon testes DNA, the three terbenzimidazole analogues exhibit flow linear dichroism properties characteristic of a minor groove-directed mode of binding to these host DNA duplexes. (iv) The apparent minor groove binding affinities of the three terbenzimidazole analogues for the d(GA4T4C)2 duplex follow a qualitatively similar hierarchy to that noted above for ligand-induced poisoning of human TOP1-namely, 5PTB > TB > 5NIBB. In the aggregate, our results suggest that DNA minor groove binding, but not DNA unwinding, is important in the poisoning of TOP1 by terbenzimidazoles.     

Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.     

Regulation of Na+/H+ exchanger gene expression. Role of a novel poly(dA.dT) element in regulation of the NHE1 promoter

In this study we examine regulation of expression of the Na+/H+ exchanger promoter in L6 and NIH 3T3 cells. We have identified a highly conserved poly(dA dT)-rich region that appears to be important in regulation of expression of the NHE1 gene. Deletion or mutation of this region results in dramatic decreases in promoter activity in both L6 and NIH 3T3 cells. In addition, DNase I footprinting experiments demonstrated that this region is protected by nuclear extracts from both cell types, and gel mobility shift assays showed that a protein or proteins specifically binds to the poly(dA dT)-rich element. Using Southwestern blotting, we determined that a 33-kDa protein binds to the poly(dA dT)-containing region. Mutations that abolished protein binding to this element diminished activity of the promoter. Insertion of the poly(dA dT)-rich element into a plasmid containing the SV40 promoter demonstrated that this element can also enhance the activity of a foreign promoter. Together, the results we have presented here show that the poly(dA dT)-rich region is important in regulation of NHE1 expression in different cell types.     

The sequence dependence of circular dichroism spectra of DNA duplexes

The three satellite DNAs of Drosophila virilis, that approximate to poly d(CAAACTA)-poly d(TAGTTTG), poly d(TAAACTA)-poly d(TAGTTTA), poly d(CAAATTA)-poly d(TAATTTG), the satellite DNA of Drosophila melanogaster that approximates to poly d(AATAT)-poly d(ATATT), the synthetic DNA duplexes, poly dG-poly dC, poly d(AT)-poly d(AT), poly d(AAT)-poly d(ATT), poly d(AAC)-poly d(GTT), poly d(TAC)-poly d(GTA) and the block copolymer d(C15A15)-d(T15G15) all have circular dichroism spectra consistent with the propositions that they have the same molecular geometry in solution and that it is the kind and frequency of nucleotide triplet sequences that determines their spectral characteristics. Poly dA-poly dT is apparently an exception.     

The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens

Although DNA is generally considered to be a poor immunogen, recent evidence suggests that DNA from various species differ in their immunological activity and that bacterial DNA can induce the in vitro proliferation of normal murine B cells. To delineate structural features of DNA associated with mitogenic activity, the response of murine lymphocytes to various natural and synthetic polynucleotides was determined. Both ss and dsDNA from two different bacterial strains were equally effective in inducing proliferation. This response was independent of adenosine methylation, since DNA from dam- Escherichia coli stimulated proliferation. Among the synthetic polymers tested, only the duplexes poly(dG).poly(dC), and poly(dG.dC) were mitogenic, while polymers containing dA, dT, or dI alone or in combination with dG and dC were inactive. The mitogenic activity of poly(dG.dC) was eliminated, however, upon substitution of rG for dG or 5medC for dC. The mitogenic activity did not require high molecular weight DNA since active polymers ranged in size from approximately 260 to 800 base pairs. In addition, E. coli DNA fragments of 50-300 and 125-600 bases were mitogenic. Together, these data suggest that the mitogenic activity of DNA is dependent on sequence-specific determinants that can be presented by synthetic DNA duplexes as well as bacterial ss and dsDNA.     

Detection of the sites of alkylation in DNA and polynucleotides by laser Raman spectroscopy

A laser Raman study of the alkylation of calf thymus DNA, poly(dG)-poly(dC) and poly(dA)-(dT) has been made using two water soluble alkylating agents: an antitumor drug, the difunctional methyl nitrogen mustard (HN2), which froms interstrand cross-links, and the dimethyl nitrogen half mustard (HN1). When an excess of the alkylating agent was used, the observed Raman frequencies due to the guanine ring modes in DNA and poly(dG)-poly(dC) changed virtually quantitatively to those of 7-methylguanosine (7-Me-Guo) showing that essentially all of the guanine bases were alkylated in the N-7 position. Furthermore, this alkylated DNA formed a stable double helical complex at neutral pH in which the alkylated guanine residues are in the keto form. No changes in the Raman bands of any of the other bases were observed in alkylated DNA. The DNA double helix, completely alkylated in at the N-7 position of guanine, melts about 35 degrees C below that of the native DNA. Upon melting, the alkylated guanine changes from the keto to the zwitterionic form.     

Probing of DNA-binding sites of Escherichia coli RecA protein utilizing 1-anilinonaphthalene-8-sulfonic acid

RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands. To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe. ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence. Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA. Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC). A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein. The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC). ATP and other nucleotides also displaced the ANS. To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest. Sequence analysis suggested that ANS binds to or near the ATP-binding region. These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand.     

Effect of Hg(II) on the spectroscopic properties of DNA bases: circular dichroism of deoxyadenosine and thymidine monomers and dimers

The addition of Hg(ClO4)2(Hg(II)) to 2'-deoxyadenosine (dA), thymidine (dT), to their respective 5'-monophosphates (dAp,dTp) as well as to the dinucleoside phosphates 2'-deoxyadenylyl-(3'-->5')-2'-deoxyadenosine (d(ApA)), 2-deoxyadenylyl-(3'-->5')thymidine(d(ApT)), thymidylyl-(3'-->5')-2'-deoxyadenosine (d(TpA)), and thymidylyl-(3'-->5')thymidine (d(TpT))--all dissolved in 0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 7--produces major alterations in the circular dichroism (CD) of the dimers but no or only small changes in the CD of the monomers. Of particular interest are the Hg(II)-induced changes in the CD of d(ApT) and d(TpA): they are strongly sequence-dependent and, within reason, progress in a "mirror"-like fashion when the concentration of Hg(II) is varied. In the absence of Hg(II), the CD of the dimers is conservative (d(TpT)), or near-conservative (d(ApA), d(ApT), d(TpA)), but becomes nonconservative upon the addition of Hg(II). The rotational strength R of the various Cotton effects of the dimers was evaluated as a function of Hg(II) concentration. Features of the CD spectra of mercurated d(ApA) and d(TpT) persist in the CD spectra of mercurated poly[d(A).(T)], but there is little obvious agreement of the CD spectra of mercurated d(ApT) and d(TpA) with the CD of mercurated poly[d(A-T).d(A-T)].     

Measuring DNA synthesis rates with [1-13C]glycine

We have devised and evaluated a stable-isotopic method for measuring DNA synthesis rates. The probe is [1-13C]-glycine that is incorporated into purines via de novo biosynthesis. The human hepatoma cell line HEP G2 was grown in medium containing [1-13C]glycine, the cells were harvested at various times, and the DNA was extracted. Following hydrolysis to the nucleosides, a reversed-phase HPLC separation was used to provide separate peaks for deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The HPLC effluent was continuously fed into a chemical reaction interface and an isotope ratio mass spectrometer (HPLC/CRI/IRMS). The isotope ratio of the CO2 produced in the CRI was used to monitor for enrichment. The cells were grown continuously for 5 days in labeled medium and also in a 1-day pulse labeling experiment where the washout of label was observed for the subsequent 9 days. As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13C/12C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1-13C]-glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer.     

Temperature effects on the structure of poly(dA).poly(dT): an X-ray fiber diffraction study

X-ray fiber diffraction of poly(dA).poly(dT) subjected to variation in the relative humidity, has allowed us to demonstrate the effects of temperature on the conformation of the polynucleotide. When the temperature of the poly(dA).poly(dT) is greater than 30 degrees C and the relative humidity near 80%, a new diffraction pattern is obtained. We observe a transition between the classical alpha B' form of poly(dA).poly(dT) and a double helical structure, B*, which remains stable at a temperature up to 70 degrees C. This new conformation of poly(dA).poly(dT) is a right-handed double helix with 11.4 nucleotide pairs per turn and a pitch of 36.7 A.     

Template specific inhibitor of mammalian DNA polymerases

A study of the inhibition of mouse cellular DNA polymerases by poly-nucleotides and their vinyl analogs is presented. Poly(dT)-directed poly(dA) synthesis by representatives of all three classes of cellular DNA polymerase could be completely inhibited by poly(9-vinyladenine), although higher concentrations were required in the case of the gamma class enzyme. Studies on the mechanism of the inhibition using the alpha class DNA polymerase and different templates showed that the enzyme activity was inhibited in all cases where base-pairing between the vinyl polymer and the template occurred; poly(9-vinyladenine) did not interfere with the replication of templates to which it does not bind. The inhibition occurred shortly after addition of poly(9-vinyladenine) to ongoing reactions, yet the enzyme was not displaced from the template - primer complex.     

The binding of echinomycin to deoxyribonucleic acid

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.