Circular dichroism calculations for double-stranded polynucleotides of repeating sequence

Optical property calculations are presented for poly(A·U), poly[(A-U)·(A-U)], poly(G·C), and poly[(G-C)·(G-C)] in RNA, B-DNA, and C-DNA conformations. An all-order classical coupled oscillator polarizability theory was used, and an effective dielectric constant of 2 was assumed. The calculated CD spectra were found to be sensitive to both geometry and sequence. Agreement with the measured CD spectra of poly(A·U), poly(G·C), and poly(dG·dC) is very good. Calculations for other sequences and geometries are less satisfactory and are particularly poor for poly[(G-C)·(G-C)] in RNA geometry and poly(A·T) in B-DNA geometry. Attempts to improve agreement with measured spectra by varying monomer properties have been only partially successful for these calculations, but they illustrate the types of changes that may prove to be necessary. Calculations using other published X-ray coordinates for certain deoxypolynucleotides of simple sequence, some of which are quite different from B-DNA coordinates, did not result in better agreement with measured spectra. Finally, the dependence of the calculated CD on chain length is examined. Results show that non-nearest neighbor interactions can be important when runs of 3 or more identical base pairs appear in a given sequence.     

The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA

Recent studies have suggested that treatment of glucocorticoid to immature growth hormone (GH)-producing cell line, MtT/S cells, dramatically induced the accumulation of GH-containing secretory granules in the cytosol and differentiated into mature GH-producing cells. However, the molecular mechanism of glucocorticoid-induced GH-containing secretory granule biogenesis in the MtT/S cells remains unknown. In the present study, we found that GH mRNA expression was facilitated by application of glucocorticoid. We artificially increased GH synthesis by transfection of green fluorescent protein-tagged GH (GH-GFP) gene. We found that the artificial elevation of GH expression in the cells did not accumulate the secretory granules in the cytosol, whereas glucocorticoid-induced the biogenesis of granules in GH-GFP-expressing MtT/S cells. We next performed DNA microarray and real-time RT-PCR analysis and found that glucocorticoid significantly altered the expression of membrane trafficking-related protein, syntaxin11 (Syx11). Immunocytochemical analysis further demonstrated that Syx11 positive structures were well colocalized with GH-containing granules in both MtT/S cells and rat anterior pituitary gland. Our findings indicate that glucocorticoid regulate the expression of Syx11 and facilitate the biogenesis and the trafficking of GH-containing granules in the MtT/S cells.     

Energetic and structural aspects of ethidium cation intercalation into DNA minihelices

The enthalpy ΔH for the intercalation of the ethidium cation (EC) into DNA minihelices can be decomposed into (1) an energy of conformational adjustment (i.e., the energy of minihelix extension and unwinding from the B-form to the intercalated form) and (2) EC minihelix intermolecular interactions. In the present study, we have focused our attention mainly on a decomposition of the energetic factors of the EC minihelix intermolecular interactions, while the essential features of the energy of conformational adjustment have been discussed in detail elsewhere by us. The structural features of the various resulting energy-minimized EC-intercalated complexes are compared with each other and the initial x-ray model structure. ΔH is estimated to be in the range of ?12.3 to ?24.0 kcal/mol. This theoretical estimate is qualitatively and quantitatively in agreement with a variety of available experimental data. The energy of conformational adjustment is an energetically unfavorable step, while the energetically favorable contribution of the EC minihelix intermolecular interactions is responsible for the overall favorable nature of the intercalation process involving the EC. On the other hand, the observed preference for intercalation into Pyr(3′–5′)Pur DNA sequences over their isomeric Pur(3′–5′)Pyr sequences is controlled by the energy of conformational adjustment and not by the EC minihelix intermolecular interaction contribution. No base-composition effect is expected at EC concentrations normally found at cellular conditions. Moreover, the structural features of the various EC-intercalated complexes are very similar regardless of minihelix base sequence or composition. These results compare favorably with available evidence. The nature of biologically preferred sites of EC binding with the minihelices is discussed.     

Ethidium bromide-dinucleotide complexes. Evidence for intercalation and sequence preferences in binding to double-stranded nucleic acids.

The solution complexes of ethidium bromide with nine different deoxydinucleotides and the four self-complementary ribodinucleoside monophosphates as well as mixtures of complementary and noncomplementary deoxydinucleotides were studied as models for the binding of the drug to DNA and RNA. Ethidium bromide forms the strongest complexes with pdC-dG and CpG and shows a definite preference for interaction with pyrimidine–purine sequence isomers. Cooperativity is observed in the binding curves of the self-complementary deoxydinucleotides pdC-dG and pdG-dC as well as the ribodinucleoside monophosphates CpG and GpC, indicating the formation of a minihelix around ethidium bromide. The role of complementarity of the nucleotide bases was evident in the visible and circular dichroism spectra of mixtures of complementary and noncomplementary dinucleotides. Nuclear magnetic resonance measurements on an ethidium bromide complex with CpG provided evidence for the intercalation model for the binding of ethidium bromide to double-stranded nucleic acids. The results also suggest that ethidium bromide may bind to various sequences on DNA and RNA with significantly different binding constants.     

A theoretical investigation on the sequence selective binding of adriamycin to double-stranded polynucleotides.

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of adriamycin (ADM) to five self-complementary double-stranded hexanucleotides. Among the two regularly alternating hexanucleotides d (TATATA)2 and d (CGCGCG)2, a stronger binding is predicted for the former. The strongest complex is computed, however, for the mixed hexanucleotide d (CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. The overall sequence preference is the result of an intricate interplay of sequence preferences of the constituents in particular of daunosamine and the 9-OH substituent. Altogether, the selective base pair recognition by adriamycin cannot be defined in terms of the two base pairs implicated in the intercalation site alone but must be expressed in terms of a triplet of base pairs.     

DNA sequence specificity of triplex-binding ligands.

We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter- and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)n or (CCT)n, but stabilizes triplexes that contain (TTC)n. In contrast, BePI slightly destabilizes the triplexes that are formed at (TC)n (CCT)n and (TTC)n. 2,7-Anthraquinone stabilizes (TC)n (CCT)n and (TTC)n, although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT)n are stabilized by the 2,7-disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT)n and neither affects the complex with (CT)n. We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C+GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C+GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues.     

Association of double-stranded DNA fragments into multistranded DNA structures.

We have previously observed that double-stranded DNA fragments containing a tract of the tandemly repeated sequence poly(CA). poly(TG) can associate in vitro to form stable complexes of low electrophoretic mobility, which are recognized with high specificity by proteins HMG1 and HMG2. The formation of such complexes has since been observed to depend on interactions of DNA with polypropylene surfaces, with the suggestion that the formation of low mobility complexes might be the result of strand dissociation followed by misaligned reassociation of the repetitive sequences. The data presented here show that at high ionic strength the interactions of DNA with polypropylene are sufficiently strong for DNA to remain bound to the polypropylene surface, which suggests that DNA might also be involved in interactions with hydrophobic molecules in vivo. Under such conditions, low-mobility complexes are found only in the material adsorbed to the polypropylene surface, and all DNA fragments are able to form low-mobility structures, whether or not they contain repetitive sequences. Preventing the separation of strands by ligating hairpin loop oligonucleotides at both ends of the fragments does not prevent the formation of low-mobility complexes. Our results suggest two different pathways for the formation of complexes. In the first, dissociation is followed by misaligned reassociation of repetitive sequences, yielding duplexes with single-stranded end regions that associate to form multimeric complexes. In the second, repetitive as well as nonrepetitive DNA molecules bound to polypropylene adopt a conformation with locally unwound regions, which allows interactions between neighboring duplexes adsorbed on the surface, resulting in the formation of low-mobility complexes.     

Supercoiling-dependent sequence specificity of mammalian DNA methyltransferase.

Negative supercoiling of substrate DNA dramatically alters the in vitro sequence specificity of mammalian DNA methyltransferase (DNA MeTase). This result suggests that in vivo site selection by DNA MeTase could be regulated by conformational information in the form of alternative secondary structures induced in DNA by local supercoiling or by the binding of specific nuclear proteins. DNA in the left-handed Z-form is shown not to be a substrate for mammalian DNA MeTase. The sensitivity of DNA MeTase to DNA structure may also make it useful as a probe for sequences which undergo supercoiling-dependent structural transitions in vitro.     

The sequence specificity of vertebrate DNA methylation.

The relative quantity of 5-methyl cytosine in vertebrate nuclear DNA shows species and tissue variation. To determine whether this is due to the action of species or cell specific DNA methylases the sequence specificity of the 5-methyl cytosine distribution in the DNA of a range of cells has been partially characterised. The pattern of methylation was found to be remarkably constant and indicates stringent evolutionary conservation of the characteristics of vertebrate DNA methylation.     

Molecular dynamics of structural transitions and intercalation in DNA

The large-scale flexibility of DNA and the intercalation of actinomycin D have been studied by computer simulation using molecular dynamics. The stretching and unwinding of B and Z forms of DNA and intercalation in B-DNA were examined through molecular dynamics simulations, and the energetics of transitions were calculated by the conformational energy minimization method. The principal results of this research are as follows: (1) A dynamic conformational pathway is presented for longitudinal stretching and unwinding of the double helix to open an intercalation site. (2) Large-scale transitions are possible in both B and Z forms of DNA through a conformationally allowed kinetic pathway. (3) The stretching and untwisting of a 5′(CG)3′ step is energetically more favorable than for a GC step in B-DNA. (4) The formation of an adjacent second cavity in B-DNA requires larger energy than the formation of the first cavity, affirming the neighbor-exclusion principle of intercalation. (5) Docking an intercalated actinomycin D in the stretched structure is shown to be geometrically and energetically feasible.     

Electrostatic components of drug-receptor recognition. I. Structural and sequence analogues of DNA polynucleotides

The electrostatic fields associated with the important biological receptor DNA have been studied by means of stereoscopic displays to investigate drug-receptor recognition processes. This revealed great differences between A- and B-type structures and enabled significant nucleotide sequence effects to be detected for the latter helix. These variations were further investigated by topological analysis of the surface potential in the two grooves of the B-DNA duplex at different radii from the helix axis. This made it possible to characterize the potential surface and to allocate curvature changes to specific atomic groupings. A general finding was that larger potential fields were found in the space encompassed by the narrow groove with strong potential gradients from the ends of the helix to the centre in both grooves. This gradient may provide a motive force for translating small molecules on the surface of a polynucleotide.     

S1 nuclease sensitivity of a double-stranded telomeric DNA sequence.

We examined structural properties of poly d(C4A2).d(T2G4), the telomeric DNA sequence of the ciliated protozoan Tetrahymena. Under conditions of high negative supercoiling, poly d(C4A2).d(T2G4) inserted in a circular plasmid vector was preferentially sensitive to digestion with S1 nuclease. Only the C4A2 strand was sensitive to first-strand S1 cutting, with a markedly skewed pattern of hypersensitive sites in tracts of either 46 or 7 tandem repeats. Linear poly d(C4A2).(T2G4) showed no preferential S1 sensitivity, no circular dichroism spectra indicative of a Z-DNA conformation, no unusual Tm, and no unusual migration in polyacrylamide gel electrophoresis. The S1 nuclease sensitivity properties are consistent with a model proposed previously for supercoiled poly d(CT).d(AG) (Pulleyblank et al., Cell 42:271-280, 1985), consisting of a double-stranded, protonated, right-handed underwound helix. We propose that this structure is shared by related telomeric sequences and may play a role in their biological recognition.     

Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity

The artificial restriction DNA cutter (ARCUT) method to cut double-stranded DNA at designated sites has been developed. The strategy at the base of this approach, which does not rely on restriction enzymes, is comprised of two stages: (i) two strands of pseudo-complementary peptide nucleic acid (pcPNA) anneal with DNA to form 'hot spots' for scission, and (ii) the Ce(IV)/EDTA complex acts as catalytic molecular scissors. The scission fragments, obtained by hydrolyzing target phosphodiester linkages, can be connected with foreign DNA using DNA ligase. The location of the scission site and the site-specificity are almost freely tunable, and there is no limitation to the size of DNA substrate. This protocol, which does not include the synthesis of pcPNA strands, takes approximately 10 d to complete. The synthesis and purification of the pcPNA, which are covered by a related protocol by the same authors, takes an additional 7 d, but pcPNA can also be ordered from custom synthesis companies if necessary.     

The sequence specificity of a mammalian DNA methylase.

The sequence specificity of an extensively purified DNA methylase preparation from Krebs II mouse ascites cells has been examined. The enzyme appears to be highly sequence dependent. Moreover the sequence distribution of cytosine residues that are methylated, bears a very close resemblance to the sequence distribution of 5'-methyl cytosine found in vivo in a wide range of vertebrate cells and is consistent with methylation of cytosines in the sequence R-Yn-C-R.     

Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA.

Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially. Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude. Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300. Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.     

Interaction of gene 5 protein of phage f1 with single and double-stranded DNA and polynucleotides

The fluorescence method was used to reveal some differences in the interaction of gene 5 protein of phage f1 with single- and double-stranded polynucleotides (DNA). The binding with the duplexes is non-cooperative and the Kapp is twice lower than that for the cooperative formation of the complex with single-stranded structures. In the complex with a double-stranded polynucleotide (DNA) the protein cover 3 nucleotide pairs. The complex dissociates with a lower concentration of salt and the contribution of the energy of nonelectrostatic interactions to the total energy of complex formation for it is lower than for the complex with single-stranded DNA. In the complex of protein with single-stranded structure the fluorescence of the tyrosine (Tyr) residues is quenched to a greater degree and their accessibility to the external quencher is lower than that of the complex with double-stranded polynucleotides (DNA). The suggestion is made that in destabilization of nucleic double helices by gene 5 protein of phage f1, a great role belongs to Tyr residues because of their high affinity to single-stranded structures and because of their different localization in the complexes with single- and double-stranded polynucleotides.     

A structural basis for S1 nuclease sensitivity of double-stranded DNA

A protonated form of a cloned simple repeating DNA sequence d(TC)n X d(GA) is detectable in equilibrium with the usual Watson-Crick base-paired form at pHs up to 7. This form is anomalously sensitive to a variety of single-strand-specific endonucleases. The observed pH dependent protection of N-7 of dG residues within the insert suggests that these residues are either Hoogsteen or reverse Hoogsteen base-paired to protonated dC residues of the polypyrimidine strand. A structure in which dA:dT Watson-Crick base pairs alternate with Hoogsteen syndG:dCH+ pairs appears to be the most stereochemically acceptable structure consistent with the chemical properties of this protonated DNA. Protonated d(TC)n X d(GA)n interacts with an anti-Z DNA antibody raised against brominated d(GC)n X d(GC)n.     

Bifunctional intercalation and sequence specificity in the binding of quinomycin and triostin antibiotics to deoxyribonucleic acid.

Quinomycin C, triostin A and triostin C are peptide antibiotics of the quinoxaline family, of which echinomycin (quinomycin A) is also a member. They all remove and reverse the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs, and the unwinding angle at I 0.01 is, in all cases, almost twice that of ethidium. Thus, as with echinomycin, they can be characterized as bifunctional intercalating agents. For the triostins this conclusion has been confirmed by measurements of changes in the viscosity of sonicated rod-like DNA fragments; the helix extension was found to be almost double that expected for a simple monofunctional intercalation process. For triostin A, further evidence for bifunctionality was derived from the cross-over point of binding isotherms to nicked circular and closed circular bacteriophage-PM2DNA. Binding curves for the interaction of quinomycin C and triostin A with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis, but triostin C was too insoluble in aqueous solution to make this method applicable. For quinomycin C the highest binding constant was found with Micrococcus lysodeikticus DNA, and its pattern of specificity among natural DNA species was broadly similar to that of echinomycin, although the binding constants were 2--6 times as large. For triostin A the highest binding constant was again found for M. lysodeikticus DNA, but the specificity pattern was quite different from that of the quinomycins. In particular, triostin A bound better to poly(dA-dT) than to the poly(dG-dC) whereas this order was reversed for quinomycin C. There was also evidence that the binding to poly(dA-dT) might be co-operative in nature. No significant interaction could be detected with poly(dA).poly(dT) or with RNA from Escherichia coli. Poly(dG).poly(dC) gave variable results, depending on the source of the polymer. The different patterns of specificity displayed by the quinomycins and triostins are tentatively ascribed to differences in their conformations in solution.