Circular dichroism of two conformations of poly[d(G-C)] induced by low pH.

Circular dichroism (CD) and UV absorption data showed that poly[d(G-C)] (at 0.09M NaCl, 0.01M Na+ (phosphate), 20 degrees C) underwent two conformational transitions upon lowering of the pH by the addition of HCl. The first transition was complete at about pH 3.0. The second transition was complete upon lowering the pH to 2.6 or upon raising the temperature, at pH 3.0, to about 40 degrees C. There was no indication of denaturation during either transition. The CD spectrum for the second acid conformation had large CD bands including a positive one at 288nm, a characteristic associated with C X C+ base-pairs. Electron microscopy showed no significant formation of condensed supramolecular aggregates corresponding to the first or second acid forms of poly[d(G-C)]. On the basis of spectral data, electron microscopy, and proton-uptake measurements, we propose models for the secondary structures that poly[d(G-C)] adopts in its two acid conformations.     

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.     

Mercury-induced transitions between right-handed and putative left-handed forms of poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)]

Poly[d(A-T).d(A-T)] and poly[d(G-C).d(G-C)], each dissolved in 0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 6.8, experience inversion of their circular dichroism (CD) spectrum subsequent to the addition of Hg(ClO4)2. Let r identical to [Hg(ClO4)2]added/[DNA-P]. The spectrum of the right-handed form of poly[d(A-T).d(A-T)] turns into that of a seemingly left-handed structure at r greater than or equal to 0.05 while a similar transition is noted with poly[d(G-C).(G-C)] at r greater than or equal to 0.12. The spectral changes are highly cooperative in the long-wavelength region above 250 nm. At r = 1.0, the spectra of the two polymers are more or less mirror images of their CD at r = 0. While most CD bands experience red-shifts upon the addition of Hg(ClO4)2, there are some that are blue-shifted. The CD changes are totally reversible when Hg(II) is removed from the nucleic acids by the addition of a strong complexing agent such as NaCN. This demonstrates that mercury keeps all base pairs in register.     

Base tilt of poly[d(A)]-poly[d(T)] and poly[d(AT)]-poly[d(AT)] in solution determined by linear dichroism

We have measured the CD, isotropic absorption, and LD of poly[d(A)]–poly[d(T)] and poly[d(AT)]–poly[d(AT)] in the vacuum-uv spectral region. The reduced dichroism (LD divided by isotropic absorption) varied as a function of wavelength and was independent of shear gradient. Thus, the bases are not perpendicular to the helix axis in solution. Since the directions of the transition dipoles are known, the orientations of the bases in the polymers can be calculated from the reduced dichroism spectra. The results show that the base normals are tilted at angles greater than 25°, with respect to the helix axis, and thymine is tilted more than adenine for both polymers. The tilt axes of adenine and thymine are not parallel, indicating a large propeller twist. Space-filling models of poly[d(A)]–poly[d(T)] and poly[(AT)]–poly[d(AT)] are built based on our results, and the conformations of the two (A + T) polymers in solution are discussed.     

Helix-destabilization of deoxyribonucleic acid and poly[d(A-T).d(A-T)] by bovine seminal ribonuclease

A ribonuclease isolated earlier from bovine seminal plasma by DNA-affinity chromatography (Ramakrishnamurti, T. and Pandit, M.W. (1983) J. Chromatogr. 260, 216-222) has now been shown by thermal denaturation studies to destabilize the double-helical structure of DNA and poly[d(A-T).d(A-T)]. Thermal denaturation profiles of DNA in the presence of the protein are much more complicated due to the denaturation of protein itself in the temperature range over which DNA predominantly melts. The protein shows relatively stronger affinity towards denatured DNA as compared to native DNA. The action of micrococcal nuclease on DNA and its complexes with ribonuclease A and bovine seminal ribonuclease indicates that both of these proteins destabilize the double-helical structure of native DNA and thereby render the DNA more sensitive to the micrococcal nuclease.     

Psi compaction of poly[d(AT)].poly[d(AT)]

The compaction of poly[d(A–T)] · poly[d(A–T)] by Co(III) is accompanied by the formation of ψ(+)- and ψ(-)-structures. The chirality of the ψ-structure depends on the Co(III) concentration, ionic strength, temperature, pH, and the chain length of the polymer. The two forms can be readily interconverted by manipulating these factors. Phase diagrams have been constructed that demonstrate the regions of stability of the enantiomers as a function of two variables, while other factors are held constant. At critical points in the phase diagram the two forms are in such unstable equilibrium that mechanical motion will cause ψ(+) ? ψ(-) interconversion. The formation of both ψ(+)- and ψ(-)-structures by the action of Co(III) on poly[d(A–T)] · poly[d(A–T)] contrasts markedly with the behavior of poly[d(G–C)] · poly[d(G–C)] in similar circumstances by forming only the ψ(+)-structure and that of native DNA to produce no ψ at all. Thus the base sequence is important in determining the structure of chirally associated DNA molecules.     

Multivalent ions are necessary for poly[d(AC).d(GT)] to assume the Z form: a CD study.

In previous work, it was shown that poly [d(AC) · d(GT)] could be forced into the Z form by strong dehydrating conditions, provided EDTA was not present. Presumably multivalent impurities were also necessary for the transition. In order to gain control over the B to Z transition for this DNA, we carefully removed all divalent contaminants from the sample and asked the obvious question: What ions are necessary for the transition under dehydrating conditions? We systematically investigated the effect of various multivalent ions. The common contaminants Ca²⁺, Mg²⁺, and Fe³⁺ will not cause the transition, but Co²⁺ and Ni²⁺ facilitate the transition, undoubtedly because of their well-known propensity to bind to purine N7. Since the transition also depends on the synergistic dehydrating action of sodium perchlorate and ethanol, we include CD spectra for the independent variations of these two factors. In addition, vacuum-uv CD spectra for the A form and various B forms of poly [d (AC) · d (GT)] are presented for the first time.     

Base tilt of B-form poly[d(G)]-poly[d(C)] and the B- and Z-conformations of poly[d(GC)]-poly[d(GC)] in solution

We have measured the CD, isotropic absorption, and linear dichroism (LD) in the vacuum-uv spectral region for the B-conformations of poly[d(G)]-poly[d(C)] and poly[d(GC)]-poly[d(GC)], and for the Z-conformation of poly[d(GC)]-poly[d(GC)] formed in 70% trifluoroethanol. The reduced dichroism (LD divided by isotropic absorption) for all conformations varied with wavelength, indicating that the bases are not perpendicular to the helix axis. Since the directions of the transition dipoles are known, the inclinations and axes of inclination of each base can be determined from the wavelength dependence of the reduced dichroism spectra. The results indicate that the base normals of the (G + C) polymers in the B- and Z-conformations are tilted at angles greater than 19° with respect to the helix axis. The guanine and cytosine bases have different inclinations, and the tilt axes are not parallel. Therefore, the bases for all the (G + C) polymer conformations studied are buckled and propeller twisted.     

The B-Z conformational transition and aggregation of poly[d(G-C)] induced by moderate concentrations of Mg(CIO4)2

Mg(ClO4)2 induces the cooperative B-to-Z transition of poly[d(G-C)]; the salt concentration at the midpoint is 0.26 M. A comparison with previous data for NaCl, MgCl2 and NaClO4 (F.M. Pohl and T.M. Jovin, J. Mol. Biol. 67 (1972) 375) indicates that Mg(ClO4)2 is more effective than would be anticipated from the simple additive effects of the Mg2+ and ClO4- ions (the ionic strengths of the respective transition points are: NaCl, 2.4; MgCl2, 2.1; NaClO4, 1.8 and Mg(ClO4)2, 0.78). These results suggest the importance of specific interactions involving ClO4-, particularly in the presence of Mg2+. The B-Z transition of poly[d(G-C)] can be monitored spectroscopically via the large hyperchromic shift at 295 nm and the inversion in the CD spectrum. The reaction is fully reversible and can be fitted by a monoexponential function with half times varying between 8 and 150 min. The observed relaxation times are strongly dependent on the concentration of Mg(ClO4)2 with a distinct maximum at the transition point, in accordance with a concerted mechanism involving only the B and Z states. As the polymer assumes the Z conformation it progressively aggregates into a gel-like precipitate, which, however, redissolves rapidly upon lowering the salt concentration. The natural DNA from Micrococcus lysodeikticus which has a high GC content of 72% is also precipitated by Mg(ClO4)2 but we do not have direct spectroscopic evidence for the involvement of the Z conformation in this phenomenon. Neither calf thymus DNA (41% GC) nor poly[d(A-T)] (0% GC) aggregates under the same conditions.     

Left-handed helical structure of poly[d(A-C)].poly[d(G-T)] studied by infrared spectroscopy

Infrared spectroscopic studies demonstrate the ability of poly[d(A-C)].poly[d(G-T)] to adopt a Z-type conformation. The Z form of the unmodified polynucleotide is induced by Ni2+ counterions and not by Na+. The B----Z equilibrium is shifted at room temperature, in the presence of 1 Ni2+/nucleotide, by an increase in the concentration of poly[d(A-C)].poly[d(G-T)]. The importance of specific binding of Ni2+ ions on the N7 site of purines in the stabilization of the Z form is also discussed.     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

Nitrogen mustard fixes the Z-conformation in poly[d(G-C)]poly[d(G-C)] and DNA]

The reactions of poly(dG-dC).poly(dG-dC) and (dG-dC)10 insert in the plasmid pGC20 with N-methyl-bis(2-chloroethyl)-amine (nitrogen mustard, HN-2) have been studied. It is shown that nitrogen mustard does not induce the B----Z transition in poly(dG-dC).poly(dG-dC), but produces fixation of the polynucleotide Z-conformation once this exists. In the case of pGC20 plasmid DNA, nitrogen mustard also fixes Z-form of the (dG-dC)-insert. The rate constant of the reaction of nitrogen mustard with guanine in the polynucleotide (k = 9,0.10(-3) min-1) is about one-third of that for the fixation of Z-form of the (dG-dC)-insert in the plasmid (k1 = 2,8.10(-2) min-1) which is attributed to a greater rate of formation of diguanyl derivative in the opposite DNA chains. It is suggested that nitrogen mustard is capable of fixing the Z-form DNA not only in vitro, but also in vivo.     

Linear dichroism demonstrates that the bases in poly[d(AC)] · poly[d(GT)] and poly[d(AG)] · poly[d(CT)] are inclined from perpendicular to the helix axis

Flow linear dichroism is used to measure specific inclinations for each of the four bases in poly[d(AC)]·;poly[d(GT)] and poly[d(AG)]·poly[d(CT)] in both the B and A forms. For the B form in solution the bases are found to have a sizable inclination. Inclination is increased in the A form, as expected. In all cases the pyrimidines are more inclined than the purines. © 1993 John Wiley & Sons, Inc.     

Right- and left-handed helixes of poly[d(A-T)].poly[d(A-T)] investigated by infrared spectroscopy

The secondary structures of double-stranded poly[d(A-T)].poly[d(A-T)] in films have been studied by IR spectroscopy with three different counterions (Na+, Cs+, and Ni2+) and a wide variety of water content conditions (relative humidity between 100 and 47%). In addition to the A-, B-, C-, and D-form spectra, a new IR spectrum has been obtained in the presence of nickel ions. The IR spectra of Ni2+-poly[d(A-T)].poly[d(A-T)] films are analyzed by comparison with previously assigned IR spectra of left-handed poly[d(G-C)].poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)], and it is possible to conclude that they reflect a Z-type structure for poly[d(A-T)].poly[d(A-T)]. The Z conformation has been favored by the high polynucleotide concentration, by the low water content of the films, and by specific interactions of the transition metal ions with the purine bases stabilized in a syn conformation. A structuration of the water hydration molecules around the double-stranded Ni2+-poly[d(A-T)].poly[d(A-T)] is shown by the presence of a strong sharp water band at 1615 cm-1.     

Changes in poly[d(T-G).d(C-A)] chirality due to Hg(II)-binding: circular dichroism (CD) studies

The conformation of poly[d(T-G).d(C-A)] in aqueous solution (0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 6.9) was studied by circular dichroism (CD) spectroscopy in the ultraviolet. The conformation of the polynucleotide, as reflected by its chiroptical signature, changes in a highly cooperative fashion in the presence of Hg(ClO4)2. The CD changes signal transitions first from the B to a modified B-state (B*), or to a non-B structure termed X, and finally to a form that is presumably Z. The alterations are totally reversible subsequent to the removal of mercury with the help of a suitable complexing agent such as sodium cyanide, indicating that mercuration does not disrupt Watson-Crick hydrogen bonding to any extent.     

Poly(dA).poly(dT) exists in an unusual conformation under physiological conditions: propidium binding to poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]

The binding of propidium to poly(dA).poly(dT) [poly(dA.dT)] and to poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]] has been compared under a variety of solution conditions by viscometric titrations, binding studies, and kinetic experiments. The binding of propidium to poly[d(A-T)2] is quite similar to its binding to calf thymus deoxyribonucleic acid (DNA). The interaction with poly(dA.dT), however, is quite unusual. The viscosity of a poly(dA.dT) solution first decreases and then increases in a titration with propidium at 18 degrees C. The viscosity of poly[d(A-T)2] shows no decrease in a similar titration. Scatchard plots for the interaction of propidium with poly(dA.dT) show the classical upward curvature for positive cooperativity. The curvature decreases as the temperature is increased in binding experiments. A van't Hoff plot of the observed binding constants yields an apparent positive enthalpy of approximately +6 kcal/mol for the propidium-poly(dA.dT) interaction. Propidium binding to poly[d(A-T)2] shows no evidence for positive cooperativity, and the enthalpy change for the reaction is approximately -9 kcal/mol. Both the magnitude of the dissociation constants and the effects of ionic strength are quite similar for the dissociation of propidium from poly(dA-T)2] and from poly[d(A-T)2], suggesting that the intercalated states are similar for the two complexes. The observed association reactions, under pseudo-first-order conditions, are quite different. Plots of the observed pseudo-first-order association rate constant vs. polymer concentration have much larger slopes for propidium binding to poly[d(A-T)2] than to poly(dA.dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

CD evidence that the alternating purine-pyrimidine sequence poly[d(A-C).d(G-T)], but not poly[d(A-T).d(A-T)], undergoes an acid-induced transition to a modified secondary conformation.

Circular dichroism and UV absorption data showed that poly[d(A-C).d(G-T)] (at 0.01M Na+ (phosphate), 20 degrees C) underwent two reversible conformational transitions upon lowering of the pH. The first transition was complete at about pH 3.9 and resulted in an acid form of the polymer that was most likely a modified, protonated duplex. The second transition occurred between pH 3.9 and 3.4 and consisted of the denaturation of this protonated duplex to the single strands. UV absorption and CD data also showed that the separated poly[d(A-C)] strand formed two acid-induced self-complexes with pKa values of 6.1 and 4.7 (at 0.01M Na+). However, neither one of these poly[d(A-C)] self-complexes was part of the acid-induced rearrangements of the duplex poly[d(A-C).d(G-T)]. Acid titration of the separated poly[d(G-T)] strand, under similar conditions, did not show the formation of any protonated poly[d(G-T)] self-complexes. In contrast to poly[d(A-C).d(G-T)], poly[d(A-T).d(A-T)] underwent only one acid-induced transition, which consisted of the denaturation of the duplex to the single strands, as the pH was lowered from 7 to 3.     

Conformational properties of poly[d(G-T)].poly[d(C-A)] and poly[d(A-T)] in low- and high-salt solutions: NMR and laser Raman analysis

³¹P- and ¹H-nmr and laser Raman spectra have been obtained for poly[d(G-T)]·[d(C-A)] and poly[d(A-T)] as a function of both temperature and salt. The ³¹P spectrum of poly[d(G-T)]·[d(C-A)] appears as a quadruplet whose resonances undergo separation upon addition of CsCl to 5.5M. ¹H-nmr measurements are assigned and reported as a function of temperature and CsCl concentration. One dimensional nuclear Overhauser effect (NOE) difference spectra are also reported for poly[d(G-T)]·[d(C-A)] at low salt. NOE enhancements between the H8 protons of the purines and the C5 protons of the pyrimidines, (H and CH₃) and between the base and H-2′,2″ protons indicate a right-handed B-DNA conformation for this polymer. The NOE patterns for the TH3 and GH1 protons in H₂O indicate a Watson–Crick hydrogen-bonding scheme. At high CsCl concentrations there are upfield shifts for selected sugar protons and the AH2 proton. In addition, laser Raman spectra for poly[d(A-T)] and poly[d(G-T)]·[d(C-A)] indicate B-type conformations in low and high CsCl, with predominantly C2′-endo sugar conformations for both polymers. Also, changes in base-ring vibrations indicate that Cs⁺ binds to O2 of thymine and possibly N3 of adenine in poly[d(G-T)]·[d(C-A)] but not in poly[d(A-T)]. Further, ¹H measurements are reported for poly[d(A-T)] as a function of temperature in high CsCl concentrations. On going to high CsCl there are selective upfield shifts, with the most dramatic being observed for TH1′. At high temperature some of the protons undergo severe changes in linewidths. Those protons that undergo the largest upfield shifts also undergo the most dramatic changes in linewidths. In particular TH1′, TCH₃, AH1′, AH2, and TH6 all undergo large changes in linewidths, whereas AH8 and all the H-2′,2″ protons remain essentially constant. The maximum linewidth occurs at the same temperature for all protons (65°C). This transition does not occur for d(G-T)·d(C-A) at 65°C or at any other temperature studied. These changes are cooperative in nature and can be rationalized as a temperature-induced equilibrium between bound and unbound Cs⁺, with duplex and single-stranded DNA. NOE measurements for poly[d(A-T)] indicate that at high Cs⁺ the polymer is in a right-handed B-conformation. Assignments and NOE effects for the low-salt ¹H spectra of poly[d(A-T)] agree with those of Assa-Munt and Kearns [(1984) Biochemistry 23 , 791–796] and provide a basis for analysis of the high Cs⁺ spectra. These results indicate that both polymers adopt a B-type conformation in both low and high salt. However, a significant variation is the ability of the phosphate backbone to adopt a repeat dependent upon the base sequence. This feature is common to poly[d(G-T)]·[d(C-A)], poly[d(A-T)], and some other pyr–pur polymers [J. S. Cohen, J. B. Wouten & C. L Chatterjee (1981) Biochemistry 20 , 3049–3055] but not poly[d(G-C)].