Chain length-dependent association of distamycin-type oligopeptides with A·T and G·C pairs in polydeoxynucleotide duplexes

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC)·poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG·dC containing sequences at moderate ionic strength and are classified as highly dA·dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG·dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG·dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

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

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

DNA binding properties of novel distamycin analogs that lack the leading amide unit at the N-terminus

First examples of distamycin (Dst) analogs which lack hydrogen bond donor or acceptor groups at the N-terminus have been synthesized. The first molecule of this series, which is a bispyrrole peptide, did not exhibit any detectable binding with double-stranded (ds) DNA. However, all other analogs did bind strongly to AT-rich sequences of ds-DNA, with the binding affinities increasing as a function of the number of repeating pyrrole carboxamide units. These results imply that a hydrogen bond donor or acceptor atom per se at the N-terminus is not a prerequisite for DNA binding in the case of pyrrole carboxamide-based Dst analogs. However, in the absence of H-bond donor or acceptor at the N-terminus, a minimum of three pyrrole carboxamide units is necessary for the onset of DNA binding. Beyond this minimum number, the binding affinity increases as a function of the number of pyrrole units, as a result of the greater availability of hydrogen bonding and van der Waals surface. Experiments with poly[d(G-C)] have shown that the presence of the N-terminus formamide group is not inevitable for GC binding of this class of molecules. The observation that the N-terminus formamide unit can be dispensed with suggests that these molecules, which are much easier to synthesize and functionalize, can be used in place of the conventional analogs of distamycin for the development of novel minor groove binders with extended sequence recognition properties.     

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.     

Interaction of the HMG1 protein with nucleic acids

Binding constants have been measured for the interaction of the protein HMG1 with native DNA, denatured DNA and a number of polynucleotides at near-physiological ionic strengths, using gel filtration and thermal denaturation. The interaction of HMG1 with DNA is shown to be noncooperative and reversible. Nucleic acids form the following series in order of increasing binding constants: poly(U) integral of poly(A) less than poly(dA) less than dsDNA integral of poly(dA) X poly(dT) integral of poly(dG) X poly(dC) much less than poly[d(A-T]) integral of ssDNA.     

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.     

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex.

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.     

Interaction of a cysteine-containing peptide with DNA

Cystine peptide dimer (Lys-Gly-Val-Cys-Val-N2H2Dns)2 with S-S bridge was synthesized and its interactions with DNA and synthetic polynucleotides have been studied by optical spectroscopy methods. By recording fluorescent titration curves we have shown that the affinity of the peptide to different synthetic polynucleotides decreases in the order: poly(dG).poly(dC) greater than poly(dA).poly(dT) greater than poly(dGC).poly(dGC). The stability of complexes to increasing concentrations of NaCl diminishes in the same order. The association constant is about 20-fold greater for peptide binding to poly(dG).poly(dC) than to poly(dA).poly(dT). By using circular dichroism and fluorescence measurements we have shown that the peptide competes for the binding sites on DNA with two minor-groove binding antibiotics--distamycin A and sybiromycin. These results have suggested that the peptide also binds in the DNA minor groove. Investigation of the interactions between such peptides and DNA may be useful for constructing ligands with combined specificity to DNA.     

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) · poly (dT), poly (dA-dT) · poly(dA-dT) and poly (dI-dC) · poly (dI-dC) but poorly, if at all, to poly (dG) · poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K_a=2.9 · 10⁵ M⁻¹); corresponding values for distamycin A are one site per 6.1 nucleotides with K_a= 11.6 · 10⁵ M⁻¹. Binding sites apparently involve predominantly A·T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3° per bound antibiotic molecule.     

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.     

Variation of DNA sequence specificity of DNA-oligopeptide binding ligands related to netropsin: imidazole-containing lexitropsins

CD binding studies of nonintercalative oligopeptides related to netropsin, named lexitropsins, have been carried out with synthetic duplex DNAs and natural DNA. While netropsin possesses a high dA.dT sequence specificity, these ligands show a progressive lowering of the ability to bind to dA.dT basepairs in DNA and a dramatic reduction of the sequence specificity seen at high salt concentration due to a replacement of pyrrole moieties by imidazoles. This variation in DNA sequence specificity of lexitropsins is mirrored in corresponding large differences in the template inactivation of poly(dA-dT).poly(dA-dT) in the RNA polymerase reaction by these drugs. The presence of imidazole permits binding of the oligopeptide to dG.dC pairs, which is most effective for the triimidazole peptide. Results at increasing salt concentration reveal, however, that a tight binding to pure dG.dC sequences does not occur. A proper sequence containing dG.dC and dA.dT pairs is supposed to be required for a higher specificity. The CD data accord well with previously reported melting studies and are in favor of recent theoretical results suggesting that the diminished AT preference may be due to an increase in the complexation energy with the dG.dC pairs.     

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.     

Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases.

The thermal stability of DNA duplexes containing deoxyinosine in a pairing position in turn with each of the four major deoxynucleotides has been investigated by measuring ultraviolet-absorbance at different temperatures. d(G2A4 X A4G2) and d(C2T4YT4C2) were prepared by the solid-phase phosphotriester method. When X is deoxyinosine, the Tm values of the duplexes are in the order Y = dC greater than dA greater than dG greater than dT greater than dU. The Tm of other duplexes containing dG, dA and dT at X were also measured. Self-complementary duplexes d(GGGAAINTTCCC) showed the same order of stability with N being dC, dA, dG and dT. Thermal stabilities of duplexes containing dG instead of dI were compared with other matched and mismatched duplexes. The Tm values of sequence isomers containing purine-pyrimidine combinations were compared. Self-complementary duplexes containing G-C and A-T in the central positions showed Tm values ca. 10 degrees higher than those containing C-G and T-A in the same positions. Thermodynamic parameters and circular dichroism spectra of these oligonucleotides were compared.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA).poly(dT) and poly(dG).poly(dC), and with triple helical poly(dA).[poly(dT)](2) and poly(dC).poly(dG).poly(dC)(+) were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA).poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG).poly(dC) and -poly(dC).poly(dG).poly(dC)(+) complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.     

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.     

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides

The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]₂ and poly(dC)·poly(dG)·poly(dC)⁺ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)⁺ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.     

Specific interactions of distamycin A and its analogs with (A-T) rich and (G-C) rich duplex regions of DNA and deoxypolynucleotides.

The specific interaction of distamycin A and analogs with DNA's and synthetic deoxypolynucleotide duplexes were studied in detail by means of circular dichroism and the data were analyzed together with viscosity results of several natural DNA's. At low ligand to nucleotide ratio the previously reported specific binding to (A-T) pairs of DNA is verified by a highly favoured interaction with (A-T)-enriched segments of distamycins containing four and five methylpyrrole carboxamide units. At higher distamycin concentration a second specific binding to (G-C) pairs most probably through hydrogen bonding is established. Viscometric results suggest a distamycin-induced local bending of the helix and could support the idea of a preferential alignment of the ligand molecule along only one strand in the groove which differs from the netropsin interaction mechanism. The possibility of an overlapping binding of the oligopeptides in the small groove is discussed.