A UV Resonant Raman Spectroscopic Study of the Interaction of Metallic Derivatives of Tetrakis(4-N-methylpyridyl)porphine with Polynucleotides

Abstract

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)₂. Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.     

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.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

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.     

Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.     

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.     

(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.     

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.     

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.     

Intercalators as probes of DNA conformation: propidium binding to alternating and non-alternating polymers containing guanine

Propidium iodide is used as a structural probe for alternating and non-alternating DNA polymers containing guanine and the results are compared to experiments with poly[d(A-T)2], poly(dA . dT) and random DNA sequences. Viscometric titrations indicate that propidium binds to all polymers and to DNA by intercalation. The binding constant and binding site size are quite similar for all alternating polymers, non-alternating polymers containing guanine and natural DNA. Poly(dA . dT) is unusual with a lower binding constant and positive cooperativity in its propidium binding isotherms. Poly(dA . dT) and poly(dG . dC) have similar salt effects but quite different temperature effects in propidium binding equilibria. Polymers and natural DNA have similar rate constants in their SDS driven dissociation reactions. The association rate constants are similar for the alternating polymers and poly(dG . dC) but are significantly reduced for poly(dA . dT). These results suggest that natural DNA, the alternating polymers, and non-alternating polymers containing guanine convert to an intercalated conformation with bound propidium in a very similar manner.     

Osmium tetroxide reactivity of DNA bases in nucleotide sequencing and probing of DNA structure.

Osmium tetroxide, 2,2'-bipyridine (Os,bipy) has been widely applied as a probe of the DNA structure. To obtain information about reactivity of DNA bases toward this probe synthetic homopolynucleotides poly(dT), poly(dC), poly(dG) and poly(dA) were treated with Os,bipy and the content of modified bases measured by stripping voltammetry and absorption spectrophotometry. After 20 hours' treatment strong modification of poly(dT) and poly(dC) and weak modification of poly(dG) were observed, while no modification was detected in poly(dA). At short incubation times under conditions close to those usually used in probing the DNA structure the extent of poly(dT) modification was more than 10 times higher than that of poly(dC). Thus, in single-stranded DNA Os,bipy reacts with T much greater than C and G. Due to the fast reaction of thymines with Os,bipy (and osmium tetroxide, pyridine) these chemicals can be applied in Maxam-Gilbert nucleotide sequencing as agents specific for thymines in single-stranded DNA.     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

Studies on the inhibition of highly purified calf thymus 8S and 7.3S DNA polymerase alpha by aphidicolin.

On activated DNA aphidicolin competitively inhibits the incorporation of dCMP by both calf thymus DNA polymerase alpha A2 and C enzymes and inhibits the incorporation of the other three deoxynucleoside monophosphates apparently non-competitively. However, aphidicolin does not inhibit the incorporation of dAMP into poly(dT) . oligo(A)10 nor does it inhibit the incorporation of dGMP into poly(dC) . oligo(dG)10, but, it does competitively inhibit the incorporation of dTMP into poly(dA) . oligo(dT)10.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite thymidine in a DNA duplex. Nonplanar alignment of epsilon dA(anti) and dT(anti) at the lesion site

Two-dimensional proton NMR studies are reported on the complementary d(C-A-T-G-T-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dT 9-mer duplex) containing 1,N6-ethenodeoxyadenosine (epsilon dA), a carcinogen-DNA adduct, positioned opposite thymidine in the center of the helix. Our NMR studies have focused on the conformation of the epsilon dA.dT 9-mer duplex at neutral pH with emphasis on defining the alignment at the dT5.epsilon dA14 lesion site. The through-space NOE distance connectivities establish that both dT5 and epsilon dA14 adopt anti glycosidic torsion angles, are directed into the interior of the helix, and stack with flanking Watson-Crick dG4.dC15 and dG6.dC13 pairs. Furthermore, the d(G4-T5-G6).d(C13-epsilon A14-C15) trinucleotide segment centered about the dT5.epsilon dA14 lesion site adopts a right-handed helical conformation in solution. Energy minimization computations were undertaken starting from six different alignments of dT5(anti) and epsilon dA14(anti) at the lesion site and were guided by distance constraints defined by lower and upper bounds estimated from NOESY data sets on the epsilon dA.dT 9-mer duplex. Two families of energy-minimized structures were identified with the dT5 displaced toward either the flanking dG4.dC15 or the dG6.dC13 base pair. These structures can be differentiated on the basis of the observed NOEs from the imino proton of dT5 to the imino proton of dG4 but not dG6 and to the amino protons of dC15 but not dC13 that were not included in the constraints data set used in energy minimization. Our NMR data are consistent with a nonplanar alignment of epsilon dA14(anti) and dT5(anti) with dT5 displaced toward the flanking dG4.dC15 base pair within the d(G4-T5-G6).d(C13-epsilon A14-C15) segment of the epsilon dA.dT 9-mer duplex.     

Experimental and theoretical study of the interaction of single-stranded DNA homopolymers and a monomethine cyanine dye: nature of specific binding.

The photophysical properties of an intercalating unsymmetrical monomethine cyanine dye and single-stranded DNA homopolymers show strong association for poly(dA) and poly(dG), but not for poly(dC) and poly(dT), as determined by several spectroscopic techniques and molecular dynamics calculations. While poly(dA) and poly(dG) appear to bind the dye as a monomer (with dramatic increase in fluorescence), poly(dC) and poly(dT) bind only very weakly, and seem to promote dye aggregation. Only in the case of poly(dA) there seems to be a unique, well defined form of intercalation, that molecular dynamics calculations suggest involve the quinoline ring between two bases, in an arrangement that should favor pi-stacking; consistently with this, the decay of the fluorescence shows a single exponential, the absorption spectrum shows a shift in the dye maximum, the fluorescence is strong, and the induced circular dichroism follows a simple pattern.     

The incorporation of wrong bases by DNA polymerase I following gamma-irradiation of DNA-like templates

The synthesis of polydeoxyribose polymers by Escherichia coli DNA polymerase I has been investigated with control and gamma-irradiated DNA-like polymer templates containing only two bases. The results show that irradiation of a poly(dA) strand leads to the incorporation of dG, whereas irradiation of poly(dC) and poly(dG) strands both lead to the incorporation of dA. Irradiation of poly(dT) does not lead to the incorporation of any wrong base. The wrong bases are incorporated into the complementary strand of the newly synthesised DNA.     

Bromodeoxyuridine mutagenesis in mammalian cells is stimulated by purine deoxyribonucleosides

The effects of purine deoxyribonucleosides on bromodeoxyurdine (BrdU) mutagenesis in Syrian hamster melanoma cells were determined. Both deoxyguanosine (dG) and deoxyadenosine (dA) were found to stimulate mutagenesis without changing the amount of BrdU in DNA. In addition, the stimulation of mutagenesis by dG and dA was suppressed by the addition of deoxycytidine (dC). These results suggest that BrdU mutagenesis involves the perturbation of dC metabolism, which perturbation is enhanced by dGTP and dATP. The mutagenic activity of dG in the absence of BrdU was tested, as was that of thymidine (dT), which we had shown previously to stimulate BrdU mutageneis. With dG alone, no increase above the spontaneous mutation frequency was detected. However, at extremely high concentration, dT in the absence of BrdU was slightly mutagenic, and the mutagenesis by dT was enhanced by dG and suppressed by dC.     

Parallel DNA double helices. II. Conformational analysis of regular helices having a second order axis of symmetry

Conformational analysis of double helices of DNA with parallel arranged sugar-phosphate chains connected by twofold symmetry has been performed. Homopolymers poly(dA).poly(dA), poly(dC).poly(dC), poly(dG).poly(dG) and poly(dT).poly(dT) were studied. For each of the homopolymers all variants of H-bond pairing were checked. The maps of closing of sugar-phosphate backbone were previously computed. By the optimization of potential energy the dihedral angles and helix parameters of relatively stable conformations of parallel stranded polynucleotides were calculated. The dependence of conformational energy on the nucleic base character and the base pair type were studied. Two main conformational regions for favourable "parallel" helix of polynucleotides were found. The former of these two regions coincide with the region of typical conformational parameters of B-DNA. On an average the conformational energy of "parallel" DNA is close to the energy of canonic "antiparallel" B-DNA.     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.