First-neighbor frequencies of satellite DNAs from Drosophila nasutoides and Pagurus pollicaris from an analysis of their circular dichroism spectra

From an analysis of their circular dichroism spectra, we find that the four (A + T)-rich satellite DNAs of Drosophila nasutoides have distributions of first-neighbor base paris that resemble those previously found for other (A + T)-rich Drosophila satellites. We also apply our spectral analysis procedure for the first time to two (G + C)-rich satellite DNAs, those from the hermit crab Pagurus pollicaris. We find that P. pollicaris satellite I cannot be accurately analyzed with our standard set of spectral components and that P. pollicaris satellite II appears to be much like the synthetic polymer poly[d(A-G-C-)·d(G-C-T)] in its first-neighbor content.     

Sequence dependence of the circular dichroism of synthetic double-stranded RNAs

We have synthesized and studied the CD spectra of five new double-stranded RNA polymers: poly[r(A-G)·r(C-U)], poly[r(A-U-C)·r(G-A-U)], poly[r(A-C-U)·r(A-G-U)], poly[r(A-A-C)·r(G-U-U)], and poly[r(A-C-C)·r(G-G-U)]. Together with previously published spectra of seven other RNA sequences, the spectra of these new sequences provide a library sufficient to approximate the spectra of all other RNA sequences by first-neighbor formulas and, in addition, give four spectra with which we may test the validity of first-neighbor approximations. (1) We find that the spectra of RNA sequence isomers are very different, but that the spectra essentially do obey first-neighbor relationships. (2) We have derived tentative first-neighbor assignments of negative bands at about 295 and 210 nm in the CD spectra. (3) A test of spectral independence shows that among the 12 polymer spectra there are at least seven significant independent spectral shapes, one less than the eight needed to give the most accurate spectral analysis of an unknown RNA sequence for its first-neighbor frequencies. (4) Spectra are calculated for RNAs of random base composition, approximating natural RNAs having complex sequences. (5) A T-matrix of spectral components assigned to the first-neighbor base pairs is derived from 10 of the spectra. This matrix allows an estimation of the CD spectrum of any other known RNA sequence or an analysis of the spectrum of an unknown sequence for its distribution of first-neighbor base-pair frequencies. (6) Test analyses of two of the synthetic polymers and of two natural RNAs set a probable limit on the accuracy of first-neighbor frequency determinations using this T-matrix. (7) Finally, we summarize in an appendix the melting temperatures for all the RNA and corresponding DNA sequences; it appears that the T_m values of both DNAs and RNAs approximately obey first-neighbor relationships.     

Rotation of periphery methylpyridine of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) and its selective binding to native and synthetic DNAs

By utilizing circular and linear dichroism, the binding mode of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) to various DNAs was studied in this work. 2-N-(methylpyridiniumyl)porphyrin(o-TMPyP), in which rotation of the periphery pyridinium ring is prevented, exhibits similar spectral properties when bound to DNA, poly[d(G-C)(2)] and poly[d(A-T)(2)], suggesting a similar binding mode. Close analysis of the spectral properties led us to conclude that o-TMPyP sits in the major groove. However, both 3-N- and 4-N-(methylpyridiniumyl)porphyrin (m- and p-TMPyP), of which the periphery pyridinium ring is free to rotate, intercalate between the basepairs of DNA and poly[d(G-C)(2)]. In the presence of poly[d(A-T)(2)], m-TMPyP exhibits a typical bisignate excitonic CD spectrum in the Soret band, while p-TMPyP shows two positive CD bands. The excitonic CD spectrum of the m-TMPyP-poly[d(A-T)(2)] complex and the positive CD band of the o-TMPyP-poly[d(A-T)(2)] complex were not affected by the presence of the minor groove binding drug, 4',6-diamidino-2-phenylindole (DAPI), indicating that this porphyrin is bound in the major groove. In contrast, two positive CD bands of the p-TMPyP-poly[d(A-T)(2)] complex altered in the presence of DAPI. From the changes in CD spectrum and other spectral properties, a few possible binding modes for p-TMPyP to poly[d(A-T)(2)] are suggested.     

The vacuum UV CD bands of repeating DNA sequences are dependent on sequence and conformation

CD spectra were obtained for eight synthetic double-stranded DNA polymers down to at least 175 nm in the vacuum uv. Three sets of sequence isomers were studied: (a) poly[d(A-C).d(G-T)] and poly[d(A-G).d(C-T)], (b) poly[d(A-C-C).d(G-G-T)] and poly[d(A-C-G).d(C-G-T)], and (c) poly[d(A).d(T)], poly[d(A-T).d(A-T)], poly[d(A-A-T).d(A-T-T)], and poly[d(A-A-T-T).d(A-A-T-T)]. There were significant differences in the CD spectra at short wavelengths among each set of sequence isomers. The (G.C)-containing sequences had the largest vacuum uv bands, which were positive and in the wavelength range of 180-191 nm. There were no large negative bands at longer wavelengths, consistent with the polymers all being in right-handed conformations. Among the set of sequences containing only A.T base pairs, poly[d(A).d(T)] had the largest vacuum uv CD band, which was at 190 nm. This CD band was not present in the spectra of the other (A.T)-rich polymers and was absent from two first-neighbor estimations of the poly[d(A).d(T)] spectrum obtained from the other three sequences. We concluded that the sequence dependence of the vacuum uv spectra of the (A.T)-rich polymers was due in part to the fact that poly[d(A).d(T)] exists in a noncanonical B conformation.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.     

A comparison of the circular dichroism spectra of synthetic DNA sequences of the homopurine . homopyrimidine and mixed purine- pyrimidine types.

We have obtained the ultraviolet circular dichroism spectra of two repeating trinucleotide DNAs, poly [d(A-G-G).d(C-C-T)] and poly[d(A-A-G).d(C-T-T)], that have all purines on one strand and all pyrimidines on the other. These spectra, together with spectra of other synthetic polymers, can be combined to give 3 first-neighbor calculations of the spectrum of poly[d(A).d(T)] and 2 first-neighbor calculations of the spectrum of poly [d(G).d(C)]. The results show (1) that first-neighbor calculations utilizing only spectra of homopurine.homopyrimidine DNA sequences are no more accurate than are similar calculations that involve spectra of mixed purine-pyrimidine sequences, demonstrating that double-stranded homopurine.homopyrimidine sequences do not obviously belong to a special class of secondary conformations, and (2) that the wavelength region above 250 nm in the CD spectra of synthetic DNAs is least predictable from first-neighbor equations, probably because this region is especially sensitive to sequence-dependent conformational differences.     

Binding of ligands to a one-dimensional heterogeneous lattice. III. Kinetic model and relaxation study of the interaction of tilorone with DNA and polynucleotides

A temperature-jump relaxation study of the interaction of tilorone with different polynucleotides and DNA has been performed. A single relaxation time, attributed to the intercalation step, has been observed in the case of poly[d(A-T)]·poly[d(A-T)], poly[d(A-C)]·poly[d(G-T)], poly[d(G-C)]·poly[d(G-C)], and poly(dG)·poly(dC). No intercalation into poly(dA)·poly(dT) occurs, and the interaction with poly(dG)·poly(dC) is different from what is observed with the other intercalating homopolymers. Refinement of the binding model is suggested from the analysis of the kinetic data. The relaxation curves obtained with DNA are well simulated based on a binding mechanism where DNA is considered a heterogeneous lattice and each type of site behaves as if it were located in the corresponding homopolymer. Poly(dA)·poly(dT) shows a unique behavior: studies of the effects of concentration and temperature indicate that tilorone acts as a probe of a process involving the polynucleotide alone. This process appears to be related to the dynamic structure of the nucleic acid and is detectable only when the bound dye is not intercalated.     

A hydrogen exchange study of the open segment in a DNA double helix

The kinetics of the hydrogen-deuterium exchange reactions of double-helical poly[d(A-T)]·poly[d(A-T)], poly(dA)·poly(dT), and constituent nucleosides (deoxyadenosine and thymidine) have been examined at various temperatures by stopped-flow ultraviolet spectrophotometry, in the spectral region 240–300 nm. The results were interpreted on the basis of a mechanism of the hydrogen exchange reaction of a helical polynucleotide, proposed by Englander and colleagues as well as by the Tsuboi and Nakanishi group. It was concluded that the rates of the base-pair opening reactions are nearly equal to one another in double-helical DNAs, irrespective of the base sequence. On the other hand, the free energy required for bringing the open segment at a particular base-pair was found to be much greater for poly(dA)·poly(dT) than for poly[d(A-T)]· poly[d(A-T)].     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Stopped-flow kinetic studies of actinomycin binding to DNAs.

Stopped-flow kinetic studies of the association of actinomycins with narural and synthetic DNA duplexes are presented. The actinomycins examined were D (C1), D lactam (in which the pentapeptide rings are closed by lactam instead of lactone linkages), X2, XObeta, and actinomine. The DNAs used included claf-thymus DNA, PM2, DNA, and two synthetic d(A-T)-lide copolymers containing 2,6-diaminopurine (DAP) in place of adenine residues, poly[d(DAP-T)]-poly[d(DAP-T)] and poly[d(DAP-A-T]-poly[d(DAP-A-T)]. Apparent equilibrium constants indicate that the DAP-containing polynucleotides bind actinomycin strongly. Comples formation of actinomycins D, D lactam, X2 and XObeta with these DNAs can be deconvoluted into five rate processes. These steps do not necessarily proceed to completion. The rates of two of these steps display a firstorder dependence on DNA concentration. The large negative entropies of activation of these steps suggest a high degree of restriction to freedom of motion on the respective transition states. The rates of the remaining three steps are independent of DNA concentration. Kinetic parameters of actinimycin binding to DNAs are presented and suggestions are made about some of the molecular evente believed to be responsible for the appearance of the five rate processes. For example, for DNA, poly[d(DAP-A-T)], and poly[d(DAP-T)], the observed order of apparent second-order rate constants, normalized to the concentration of actinomycin binding sites, suggests that binding of the antibiotic occurs most rapidly at binding sites (G-C of d DAT-T) near d(A-T) base pairs, where weakening of the double-helical conformation requires the least energy. Results obtained from studies of actinomycin D binding to heat-denatured poly[d(DAP-A-T)] and of actinomine and actinomycin D lactam binding to DNA suggest that the slow rate processes are related to an actinomycyl-pentapeptide-induced unwinding of the sugar-phosphate backbone of DNA accompanying insertion of the cyclic peptides into DNA.     

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.     

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)].     

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.     

Characteristics and variations of B-type DNA conformations in solution: a quantitative analysis of Raman band intensities of eight DNAs

Raman spectra were obtained from four bacterial DNAs varying in GC content and four periodic DNA polymers in 0.1 M NaCl at 25 degrees C. A curve fitting procedure was employed to quantify and compare Raman band characteristics (peak location, height, and width) from 400 to 1600 cm-1. This procedure enabled us to determine the minimum number of Raman bands in regions with overlapping peaks. Quantitative comparison of the Raman bands of the eight DNAs provided several new results. All of the DNAs examined required bands near 809 (+/- 7) and 835 (+/- 5) cm-1 to accurately reproduce the experimental spectra. Since bands at these frequencies are associated with A-family and B-family conformations, respectively, this result indicates that all DNAs in solution have a mixture of conformations on the time scale of the Raman scattering process. Band characteristics in the 800-850-cm-1 region exhibited some dependence on CG content and base pair sequence. As previously noted by Thomas and Peticolas [Thomas, G. A., & Peticolas, W. L. (1983) J. Am. Chem. Soc. 105, 993], the poly[d(A)].poly[d(T)] spectra were qualitatively distinct in this region. The A-family band is clearly observed at 816 cm-1. The intensity of this band and that of the B-family band at 841 cm-1 were similar, however, to intensities in the natural DNA spectra. Three bands at 811, 823, and 841 cm-1 were required to reproduce the 800-850-cm-1 region of the poly[d(A-T)].poly[d(A-T)] spectra. This may indicate the presence of three backbone conformations in this DNA polymer. Analysis of intensity vs. GC content for 42 Raman bands confirmed previous assignments of base and backbone vibrations and provided additional information on a number of bands.     

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.     

Equilibrium binding of benzo[a]pyrene tetrol to synthetic polynucleotides: sequence selectivity, thermodynamic properties, and ionic strength dependence

We have investigated the equilibrium binding of racemic 7r,8t,9t,10c-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene to the double-stranded, synthetic polynucleotides poly[d(A-T)], poly[d(G-C)], and poly[d(G-m5C)] at low binding ratios. Difference absorption spectroscopy shows a 10-nm red shift for binding to poly[d(A-T)] and an 11-nm red shift for binding to either poly[d(G-C)] or poly[d(G-m5C)]. The value of delta epsilon for binding is approximately the same for all three hydrocarbon-polynucleotide complexes. Binding of this neutral polycyclic aromatic hydrocarbon derivative to these polynucleotides is dependent upon ionic strength and temperature. Analysis of complex formation employing polyelectrolyte theory shows a greater release of counterions associated with binding to poly[d(A-T)] than with the other two polynucleotides (0.5 and ca. 0.36, respectively). Thus, sequence-selective binding of this hydrocarbon in DNA would be expected to change depending on salt concentration. The temperature dependence of binding was studied at 100 mM Na+ where the equilibrium binding constants for poly[d(A-T)] and poly[d(G-m5C)] are roughly equivalent and 6-fold greater than the binding affinity for poly[d(G-C)]. The binding to poly[d(A-T)] and poly[d(G-C)] is characterized by a delta H omicron = -7.0 kcal/mol, and the large difference in affinity constants arises from differences in negative entropic contributions. Formation of hydrocarbon-poly[d(G-m5C)] complexes is accompanied by a delta H = -9.1 kcal/mol. However, the affinity for poly[d-(G-m5C)] is the same as that for poly[d(A-T)] due to the much more negative entropy associated with binding to poly[d(G-m5C)].     

CD of the synthetic RNA duplexes poly[r(A-T)] and poly[r(A-U)] in salt and ethanolic solutions.

Synthetic RNA poly[r(A-T)] has been synthesized and its CD spectral properties compared to those of poly[r(A-U)], poly[d(A-T)], and poly[d(A-U)] in various salt and ethanolic solutions. The CD spectra of poly[r(A-T)] in an aqueous buffer and of poly[d(A-T)] in 70.8% v/v ethanol are very similar, suggesting that they both adopt the same A conformation. On the other hand, the CD spectra of poly[r(A-T)] and of poly[r(A-U)] differ in aqueous, and even more so in ethanolic, solutions. We have recently observed a two-state salt-induced isomerization of poly[r(A-U)] into chiral condensates, perhaps of Z-RNA [M. Vorlícková, J. Kypr, and T. M. Jovin, (1988) Biopolymers 27, 351-354]. It is shown here that poly[r(A-T)] does not undergo this isomerization. Both the changes in secondary structure and tendency to aggregation are different for poly[r(A-T)] and poly[r(A-U)] in aqueous salt solutions. In most cases, the CD spectrum of poly[r(A-U)] shows little modification of its CD spectrum unless the polymer denatures or aggregates, whereas poly[r(A-T)] displays noncooperative alterations in its CD spectrum and a reduced tendency to aggregation. At high NaCl concentrations, poly[r(A-T)] and poly[r(A-U)] condense into psi(-) and psi(+) structures, respectively, indicating that the type of aggregation is dictated by the polynucleotide chemical structure and the corresponding differences in conformational properties.     

CD of homopolymer DNA-RNA hybrid duplexes and triplexes containing A-T or A-U base pairs.

CD spectra and difference-CD spectra of (a) two DNA X RNA hybrid duplexes (poly[r(A) X d(U)] and poly[r(A) X d(T)]) and (b) three hybrid triplexes (poly-[d(T) X r(A) X d(T)], poly[r(U) X d(A) X r(U)], and poly[r(T) X d(A) X r(T)]) were obtained and compared with CD spectra of six A X U- and A X T-containing duplex and triplex RNAs and DNAs. We found that the CD spectra of the homopolymer duplexes above 260 nm were correlated with the type of base pair present (A-U or A-T) and could be interpreted as the sum of the CD contributions of the single strands plus a contribution due to base pairing. The spectra of the duplexes below 235 nm were related to the polypurine strands present (poly-[r(A)] or poly[d(A)]). We interpret the CD intensity in the intermediate 255-235 nm region of these spectra to be mainly due to stacking of the constituent polypurine strands. Three of the five hybrids (poly[r(A) X d(U)], poly[r(A) X d(T)], and poly[d(T) X r(A) X d(T)]) were found to have heteronomous conformations, while poly[r(U) X d(A) X r(U)] was found to be the most A-like and poly[r(T) X d(A) X r(T)], the least A-like.     

The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Abstract

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly(d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)₂₀, d(pA)₇, and d(pT)₇). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometrics depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 ± 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)) (with the first endpoints giving n = 4.1, 4.0, 4.0, and 4.1 ± 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non- interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol Struct, and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex.

We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A- and/or T-containing DNAs, Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20°C, appeared to interact with one another in much the same manner as in the free polymers at 50°C. Finally, we found that the protein tyrosyl CD band at 228.5 nm decreased 39-42% when the protein bound to poly[d(A)] or poly[d(T)], but this band decreased no more than 9% when the gene 5 protein bound to short A- or T-containing oligomers. Thus, at least one tyrosyl residue has a significantly altered optical activity only when the DNA substrate is long enough either to cause a transition to a different protein conformation or to allow additional protein-protein contacts between adjacent helical turns of the DNA-protein complex.