Poly(dG).poly(dC) at neutral and alkaline pH: the formation of triple stranded poly(dG).poly(dG).poly(dC).

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.     

Vacuum UV CD of the low-salt Z-forms of poly(rG-dC).poly(rG-dC), and poly(dG-m5dC).poly(dG-m5dC)

The vacuum CD spectra of poly(rG-dC)·poly(rG-dC) and poly(dG-m⁵dC)·poly(dG-m⁵dC) have been obtained for the low-salt Z-conformations of both polymers. The spectra are very similar to those for the high-salt Z-forms. This behavior is consistent with the suggestion that the low- and high-salt Z-forms are comprised of different proportions of Z_I- and Z_II-conformations.     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.     

The crystal structure of d(G-G-G-G-C-C-C-C). A model for poly(dG).poly(dC)

The structure of the DNA oligomer d(G-G-G-G-C-C-C-C) has been determined at a resolution of 2.5 A by single-crystal X-ray methods. There are two strands in the asymmetric unit, and these coil about each other to form a right-handed double-helix of the A-type with Watson-Crick hydrogen bonds between base-pairs. The helix has a shallow minor groove and a deep, water-filled major groove; almost all exposed functional groups on the DNA are hydrated, and 106 ordered solvent molecules have been found. The two d(G-G-G-G).d(C-C-C-C) segments in the octamer exhibit similar and uniform structures, but there is a slight discontinuity at the GpC step between them. A recurring feature of the structure is the overlap of adjacent guanine bases in each GpG step, with the five-membered ring of one guanine stacking on the six-membered ring of its neighbour. There is little or no overlap between adjacent cytosine rings. Conformational parameters for these GpG steps are compared with those from other single-crystal X-ray analyses. In general, GpG steps exhibit high slide, low roll and variable twist. Models for poly(dG).poly(dC) were generated by applying a simple rotation and translation to each of the unmodified d(G-G-G-G).d(C-C-C-C) units. Detailed features of these models are shown to be compatible with various assays of poly(dG).poly(dC) in solution, and are useful in understanding the polymorphic behaviour of this sequence under a variety of experimental conditions.     

A circular dichroism study of poly dG, poly dC, and poly dG:dC

We have measured the ultraviolet circular dichroism spectra of oligo d(pG)₅, poly dN AcG, poly dI, poly dC, two samples of poly dG, and four samples containing double-stranded poly dG:dC. We find that oligo d(pG)₅ and poly dG exist in self-complexed forms as well as in single-stranded forms. Unlike the self-complexed form of poly dG, the single-stranded form of poly dG can hydrogen-bond with single-stranded poly dC. We present spectral data for double-stranded poly dG:dC, which can be used to help characterize poly dG:dC preparations and which provide a basis for resolving discrepancies among other reported poly dG:dC spectra.     

The molecular electrostatic potential and steric accessibility of poly (dI.dC). Comparison with poly (dG.dC)

The influence of the amino group of guanine on the molecular electrostatic potential and the accessibility to reactive sites of B-DNA is investigated by comparing the two model double helices poly (dI.dC) and poly (dG.dC). The calculations clarify the “disruptive” role of the guanine amino group on nucleic acid-polypeptide interactions.     

Nucleosome reconstitution on plasmid-inserted poly(dA) . poly(dT).

Chromatin was reconstituted from core histones and recombinant plasmid DNAs carrying poly(dA) . poly(dT) inserts of various lengths. A 97-bp insert was found to occupy discrete and regularly-spaced positions on the edges of the nucleosome. This insert cannot, however, be entirely included due to a block in the center of the particle. In contrast, nucleosomes reconstitute on a shorter 20-bp insert. In this case, the insert shows a marked preference for the edges of the particle. Possible structural and physiological implications of these observations are discussed.     

Protonated polynucleotides structures - 23. The acid-base hysteresis of poly(dG).poly(dC).

The large hysteresis observed during the acid-base titration of poly(dG). poly (dC) was studied by CD and potentiometric scanning curves. Intermediate scanning loops as well as the equilibrium and metastable branches of the hysteresis loop have been determined. The potentiometric titrations showed, however, that the various complexes were not discrete entities, but were linked in "polycomplexes" as had been already suggested. This prevented a thermodynamic study of the system. The acid-base titration was further investigated as a function of ionic strength and temperature. The pK's showed considerably lower ionic strength dependence than observed for polyribonucleotide complexes. The thermal transitions permitted to establish the relative stabilities of the various complexes between pH 2.5 and pH 12.0.     

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.     

High-resolution A-DNA crystal structures of d(AGGGGCCCCT). An A-DNA model of poly(dG) x poly(dC).

A-DNA conformation is favored by guanine-rich sequences, such as (dG)n x (dC)n, or under low-humidity conditions. Earlier A-DNA crystal structures revealed some conformational variations which may be the result of sequence-dependent effects and/or crystal packing forces. Here we report the high-resolution crystal structure of d(AGGGGCCCCT) in two crystal forms (either in the P212121 or the P6122 space group) to gain insights into the conformation and dynamics of the (dG)n x (dC)n sequence. The P212121 form has been analyzed using data to 1.1 A resolution by the anisotropic temperature factor refinement procedure of the SHELX97 program. Such analysis affords us with the detailed geometric, conformational and motional property of an A-DNA structure. The backbone torsional angles fall in a narrow range, except for the alpha/gamma angles which have two distinct combinations (gauche-/gauche+ or trans/trans). An A-DNA model of poly(dG) x poly(dC) has been constructed using the conformational parameters derived from the crystal structure of the P212121 form. In the crystal structure of the P6122 space group, the central eight base pairs of the decamer adopt A-DNA conformation with the two terminal nucleotides flipped out to form base pairs with the neighboring nucleotides. Comparison of the A-DNA structure of the same sequence from two different crystal forms, reinforced the conclusion that molecules crystallized in the same space group have a more similar conformation, whereas the same molecule crystallized in different space groups has different (local) conformations.     

Description of base motion and role of excitations in the melting of poly(dG).poly(dC).

We study the contribution of various vibrational modes to the melting of poly(dG).poly(dC). We find that the principal contribution comes from the H-bond breathing modes that have been observed in Raman scattering and that we have associated with helix melting. We show the softening of these modes on approach to melting in agreement with the observed behavior. We also describe the contribution to melting from base rotation modes that others have suggested are important in melting.     

Protonated forms of poly[d(G-C)] and poly(dG).poly(dC) and their interaction with berberine

The pH -induced structural changes on the conformation of homo- and hetero-polymers of guanosine-citydine (G.C) sequences were investigated using spectrophotometric and circular dichroic techniques. At pH 3.40, 10 mM [Na(+)] and 10 degrees C both polynucleotides adopted a unique and stable structural conformation different from their respective B-form structures. The protonated hetero-polymer is established as left-handed structure with Hoogsteen base pairing (H(L)-form) while the homo-polymer favored Watson-Crick base pairing with different stacking arrangements from that of B-form structure as evident from thermal melting and circular dichroic studies. The interaction of berberine, a naturally occurring protoberberine group of plant alkaloid, with the protonated structures was studied using various biophysical techniques. Binding of berberine to the H(L)-form structure resulted in intrinsic circular dichroic changes and generation of extrinsic circular dichroic bands with opposite sign and magnitude compared to its B-form structure while with the homo-polymer of G.C no such reversal of extrinsic circular dichroic bands was seen indicating different stacking arrangement of berberine at the interaction site. Scatchard analysis of the binding data, however, indicated non-cooperative binding to both the protonated forms similar to that of their respective B-form structure. Fluorescence spectral studies, on the other hand, showed remarkable increase in the intrinsic fluorescence of the alkaloid in presence of the protonated forms compared to their respective B-form structure. These results suggest that berberine could be used as a probe to detect the alteration of structural handedness due to protonation and may potentiate its use in regulatory roles for biological functions.     

Protonated polynucleotide structures, 20. Interaction between poly(dG)-poly(dC) and poly(rC).1.

A study of the interaction between poly(dG)-poly(dC) and poly(rC) demonstrates that, at neutral pH and high ionic strength, there is replacement of the dC strand by poly(rC). At acid pH, formation of a triple-stranded complex which equally may involve the replacement phenomenon is observed. There is no evidence for interaction at neutral pH between poly(dG)-poly(dC) and oligo(rC), while a three-stranded complex is formed at acid pH. These data are consistent with the studies of comparative stabilities of double stranded deoxy or ribo polymers and deoxy-ribo hybrids.     

Protonated polynucleotides structures - 22.CD study of the acid-base titration of poly(dG).poly(dC)

The acid-base titration (pH 8 --> pH 2.5 --> pH 8) of eleven mixing curve samples of the poly(dG) plus poly(dC) system has been performed in 0.15 M NaCl. Upon protonation, poly(dG).poly(dC) gives rise to an acid complex, in various amounts according to the origin of the sample. We have established that the hysteresis of the acid-base titration is due to the non-reversible formation of an acid complex, and the liberation of the homopolymers at the end of the acid titration and during the base titration: the homopolymer mixtures remain stable up to pH 7. A 1G:1C stoichiometry appears to be the most probable for the acid complex, a 1G:2C stoichiometry, as found in poly(C(+)).poly(I).poly(C) or poly(C(+)).poly(G).poly(C), cannot be rejected. In the course of this study, evidence has been found that the structural consequences of protonation could be similar for both double stranded poly(dG).poly(dC) and G-C rich DNA's: 1) protonation starts near pH 6, dissociation of the acid complex of poly(dG).poly(dC) and of protonated DNA take place at pH 3; 2) the CD spectrum computed for the acid polymer complex displays a positive peak at 255 nm as found in the acid spectra of DNA's; 3) double stranded poly(dG).poly(dC) embedded in triple-stranded poly(dG).poly(dG).poly(dC) should be in the A-form and appears to be prevented from the proton induced conformational change. The neutral triple stranded poly(dG).poly(dG).poly(dC) appears therefore responsible, although indirectly, for the complexity and variability of the acid titration of poly(dG).poly(dC) samples.     

Reversible helix/coil transitions of left-handed Z-DNA structures. Comparison of the thermodynamic properties of poly(dG).poly(dC), poly[d(G-C)].poly[d(G-C)], and poly(dG-m5dC).poly(dG-m5dC)

In contrast to poly(dG).poly(dC), which remains in the B-DNA conformation under all experimental conditions the polynucleotides with the strictly alternating guanine/cytosine or guanine/5'-methylcytosine sequences can change from the classical right-handed B-DNA structure to the left-handed Z-DNA structure when certain experimental conditions such as ionic strength or solvent composition are fulfilled. Up to now the investigation of the helix/coil transition of left-handed DNA structures was not possible because the transition temperature exceeds 98 degrees C. By applying moderate external pressure to the surface of the aqueous polymer solution in the sample cell the boiling point of the solvent water is shifted up the temperature scale without shifting the transition temperature, so that we can measure the helix/coil transition of the polynucleotides at all experimental conditions applied. It can thus be shown that the Z-DNA/coil transition is cooperative and reversible. The Tm is 125 degrees C for poly(dG-m5dC).poly(dG-m5dC) in 2mM Mg2+, 50mM Na+, pH 7.2 and 115 degrees c for poly[d(G-C)].poly[d(G-C)] in 3.04M Na+. The transition enthalpy per base pair was determined by the help of an adiabatic scanning microcalorimeter.     

Calculated microwave absorption of double-helical B-conformation poly(dG)·poly(dC)

We have estimated the absorption of microwave radiation by the four acoustic modes of poly(dG)·poly(dC) in B conformation. The eigenvectors used in this calculation are those obtained from a refined normal coordinate analysis by fitting the velocity of the longitudinal acoustic mode to that observed by Brillouin scattering. The acoustic modes couple to the radiation field when translational invariance is broken such as for finite segments of helix and as may be the case in vivo. Calculations for various helix lengths show that the estimated absorption is greater than that of water by at least two to three orders of magnitude in some cases.     

Salt induced B----A transition of poly(dG).poly(dC) and the stabilization of A form by its methylation.

Raman spectra of poly(dG).poly(dC) have been observed in aqueous solutions at various ionic strengths, [NaCl] = 0.03 to 4 M, and at different temperatures, 10 to 60 degrees C. At 30 degrees C, and at [NaCl] = 0.03 M, it was found to have a B-form (with O4'endo-anti guanosine and C2'endo-anti cytidine), whereas, at [NaCl] = 4 M, an A form (with C3'endo-anti guanosine and C3'endo-anti cytidine). At 30 degrees C and [NaCl] = 1 M, namely at an intermediate state, a fraction of this molecules was considered to have a "heteronomous A" form (with O4'endo-anti guanosine and C3' endo-anti cytidine). At 60 degrees C and [NaCl] = 1 M, it assumes the B form, and at 10 degrees C and [NaCl] = 1 M, the A form. Cytosine-5-methylation was found to cause a marked stabilization of the A form. Even at [NaCl] = 0.1 M (at 30 degrees C), a substantial portion of poly(dG).poly(dm5C) was found to have a heteronomous form, in which the dG atrand is in the B form and the dC an A form; it never assumes a complete B form.     

500-MHz 1H NMR study of poly(dG).poly(dC) in solution using one-dimensional nuclear Overhauser effect

Secondary structures of poly(dG).poly(dC) and poly(dG).poly(dm5C) in solution are determined by nuclear Overhauser effect (NOE) measurements on GH8-deuterated and -nondeuterated DNAs with low presaturation pulse lengths (10-25 ms) and low-power and prolonged accumulations in the range of 50,000-72,000 scans. Under these conditions, the NOE difference spectra were free from diffusion. Primary NOEs between base protons GH8/CH6 and sugar protons H1', H2'/H2', and H3' suggest that in poly(dG).poly(dC) both guanine and cytosine nucleotides adopt a C3'-endo, low anti X = 200-220 degrees conformation. Computer modeling of the NOE data enable identification for the first time, in terms of the geometry of the nucleotide repeat, handedness, and helix geometry, of the structure of poly(dG).poly(dC) to be the A form, and the derived structure for the polymer duplex is very close to the single crystal structure of the double-helical d-GGGGCCCC [McCall, M., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 183, 385-396]. Similar nuclear Overhauser effect data on poly(dG).poly(dm5C) revealed that G and m5C adopt a C2'endo, anti X = 240-260 degrees conformation, which indicates that this DNA exhibits the B form in solution. In summary, the results presented in this paper demonstrate that methylation of cytosines in poly(dG).poly(dC) causes A----B transition in the molecule.