The binding of fd gene 5 protein to polydeoxynucleotides: evidence from CD measurements for two binding modes

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 stoichiometries 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 degrees C, appeared to interact with one another in much the same manner as in the free polymers at 50 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of the Binding of fd Gene 5 Protein to Polydeoxyribonucleotides

Abstract

The long-wavelength circular dichroism (CD) changes induced by binding of fd gene 5 protein to the alternating DNA sequences poly[d(A-C)] and poly [d(C-T)] were similar to those induced by the protein complexed with the homopolymers poly[d(A)], poly[d(C)], and poly[d(T)]. The fd gene 5 protein showed different binding affinities for the various polymers. The affinity for the alternating sequences was not compositionally weighted with respect to the affinities for the homopolymers, indicating that both base composition and base sequence of the template are important for the binding of fd gene 5 protein.     

Specificity of the binding of fd gene 5 protein to polydeoxyribonucleotides

The long-wavelength circular dichroism (CD) changes induced by binding of fd gene 5 protein to the alternating DNA sequences poly[d(A-C)] and poly[d(C-T)] were similar to those induced by the protein complexed with the homopolymers poly[d(A)], poly[d(C)], and poly[d(T)]. The fd gene 5 protein showed different binding affinities for the various polymers. The affinity for the alternating sequences was not compositionally weighted with respect to the affinities for the homopolymers, indicating that both base composition and base sequence of the template are important for the binding of fd gene 5 protein.     

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.     

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.     

Binding and reversible denaturation of double-stranded DNA by Ff gene 5 protein

The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+).     

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.     

Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein · nucleic acid complexes

We used a mutant gene 5 protein (g5p) to assign and interpret overlapping CD bands of protein · nucleic acid complexes. The analysis of overlapping protein and nucleic acid CD bands is a common challenge for CD spectroscopists, since both components of the complex may change upon binding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mutant gene 5 protein that has an insignificant change in tyrosine optical activity at 229 nm upon binding to nucleic acids. We have studied the interactions of the mutant Y34F g5p (Tyr-34 substituted with phenylalanine) with poly[r(A)], poly[d(A)], and fd single-stranded DNA (ssDNA). Our results showed the following: (1) The 205–300 nm spectrum of poly[r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentially the sum of the spectra of poly[r(A)] at a high temperature plus the spectrum of the free protein, except for a minor negative band at 257 nm. (2) The spectra of poly[d(A)] and fd ssDNA saturated with the mutant protein at a P/N = 0.25, minus the spectra of the free nucleic acids at a high temperature, also essentially equaled the spectrum of the free protein in the 205–245 nm region. (3) While the overall secondary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of individual aromatic residues. (4) Nucleic acids complexed with the g5p are unstacked (as if heated) and (in the cases of the DNAs) perturbed as if part of a dehydrated double-stranded DNA. (5) Difference spectra revealed regions of the spectrum specific for the particular nucleic acid, the protein, and whether g5p was bound to DNA or RNA. © 1997 John Wiley and Sons, Inc. Biopoly 42: 337–348, 1997     

Electron microscopy of a eukaryotic single-stranded DNA binding protein-DNA complex

The single-stranded DNA binding protein of Ustilago maydis decreases the contour length of φX174 DNA. When DNA complexes were prepared with subsaturating amounts of the protein, its distribution on the DNA was markedly non-random, indicating a high degree of co-operativity in its binding to single-stranded DNA. The analagous Escherichia coli, Salmonella typhimurium and bacteriophage T7 binding proteins also reduced DNA contour lengths to a similar extent, whereas the bacteriophage T4 gene 32 protein, as shown previously, increased the contour length. Despite the fact that the U. maydis protein efficiently denatures poly[d(A-T) · d(A-T)], it appears to initiate denaturation of native bacteriophage λ DNA rather inefficiently.     

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.     

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.     

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)].     

Binding of IKe gene 5 protein to polynucleotides. Fluorescence binding experiments of IKe gene 5 protein and mutual cooperativity of IKe and M13 gene 5 proteins

Fluorescence studies of the binding of IKe gene 5 protein to various polynucleotides were performed to obtain insight into the question as to what extent the binding characteristics of the gene 5 proteins of the IKe and M13 phages resemble and/or differ from each other. The fluorescence of IKe gene 5 protein is quenched 60% upon binding to most polynucleotides. At moderate salt concentrations, i.e., below 1 M salt, the binding stoichiometry is 4.0 +/- 0.5 nucleotides per IKe gene 5 protein monomer. The affinity of the protein for homopolynucleotides depends strongly on sugar and base type; in order of increasing affinities we find poly(rC) less than poly(dA) less than poly(rA) less than poly(dI) less than poly(rU) less than poly(dU) less than poly(dT). For most polynucleotides studied, the affinity depends linearly on the salt concentration: [d log (Kint omega)]/(d log [M+]) = -3. The binding is highly cooperative. The cooperativity parameter omega, as deduced from protein titration curves, is 300 +/- 150 and appears independent of the type of polynucleotide studied. Estimation of this binding parameter from salt titrations of gene 5 protein-polynucleotide complexes results in systematically higher values. A comparison of the binding data of the IKe and M13 gene 5 proteins shows that the fluorescence quenching, stoichiometry, order of binding affinities, and cooperativity in the binding are similar for both proteins. From this it is concluded that at least the DNA binding grooves of both proteins must show a close resemblance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH相似文献   

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.     

Binding geometries of triple helix selective benzopyrido [4,3-b]indole ligands complexed with double- and triple-helical polynucleotides

The binding modes of three benzopyrido [4,3-b]indole derivatives (and one benzo[-f]pyrido [4-3b] quinoxaline derivative) with respect to double helical poly(dA) · poly(dT) and poly[d(A-T)]₂ and triple-helical poly(dA) · 2poly(dT) have been investigated using linear dichroism (LD) and CD: (I) 3-methoxy-11-amino-BePI where BePI = (7H-8-methyl-benzo[e]pyrido [4,3-b]indole), (II) 3-methoxy-11-[(3′-amino) propylamino]-BePI, (III) 3-methoxy-7-[(3′-diethylamino)propylamino] BgPI where BgPI = (benzo[g]pyrido[4,3-b]indole), and (IV) 3-methoxy-11-[(3′-amino)propylamino] B f P Q where B f P Q = {benzo[-f]pyrido[4-3b]quinoxaline}. The magnitudes of the reduced LD of the electronic transitions of the polynucleotide bases and of the bound ligands are generally very similar, suggesting an orientation of the plane of the ligands' fused-ring systems preferentially perpendicular to the helix axis. The LD results suggest that all of the ligands are intercalated for all three polynucleotides. The induced CD spectrum of the BePI chromophore in the (II-BePI)-poly[d(A-T)]₂ complex is almost a mirror image of that for the (I-BePI)-poly(dA) · poly(dT) and (I-BePI)-poly(dA) · 2poly(dT) complexes, suggesting an antisymmetric orientation of the BePI moiety upon intercalation in poly[d(A-T)]₂ compared to the other polynucleotides. The induced CD of I-BePI bound to poly(dA) · 2poly(dT) suggests a geometry that is intermediate between that of its other two complexes. The concluded intercalative binding as well as the conformational variations between the different BePI complexes are of interest in relation to the fact that BePI derivatives are triplex stabilizers. © 1997 John Wiley & Sons, Inc. Biopoly 42: 101–111, 1997     

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.     

Spectroscopic studies of 9-hydroxyellipticine binding to DNA

The binding of 9-hydroxyellipticine to calf thymus DNA, poly[d(A-T)]₂, and poly-[d(G-C)]₂ has been studied in detail by means of CD, linear dichroism, resonance light scattering, and molecular dynamics. The transition moment polarizations of 9-hydroxyelliptiycine were determined in polyvinyl alcohol stretched film. Spectroscopic solution studies of the DNA/drug complex are combined with theoretical CD calculations using the final 50 ps of a series of molecular dynamics simulations as input. The spectroscopic data shows 9-hydroxyellipticine to adopt two main binding modes, one intercalative and the other a stacked binding mode involving the formation of drug oligomers in the DNA major groove. Analysis of the intercalated binding mode in poly[d(A-T)]₂ suggests the 9-hydroxyellipticine hydroxyl group lies in the minor groove and hydrogen bonds to water with the pyridine ring protruding into the major groove. The stacked binding mode was examined using resonance light scattering and it was concluded that the drug was forming small oligomer stacks rather than extended aggregates. Reduced linear dichroism measurements suggested a binding geometry that precluded a minor groove binding mode where the plane of the drug makes a 45° angle with the plane of the bases. Thus it was concluded that the drug stacks in the major groove. No obvious differences in the mode of binding of 9-hydroxyellipticine were observed between different DNA sequences; however, the stacked binding mode appeared to be more favorable for calf thymus DNA and poly[d(G-C)]₂ than for poly[d(A-T)]₂, an observation that could be explained by the slightly greater steric hindrance of the poly[d(A-T)]₂ major groove. A strong concentration dependence was observed for the two binding modes where intercalation is favored at very low drug load, with stacking interactions becoming more prominent as the drug concentration is increased. Even at DNA : drug mixing ratios of 70:1 the stacked binding mode was still important for GC-rich DNAs. © 1998 John Wiley & Sons, Inc. Biopoly 46: 127–143, 1998     

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.     

DNA binding proteins from Tetrahymena thermophila

We have used DNA-cellulose chromatography to isolate single-strand binding proteins from Tetrahymena thermophila. Three major proteins which bind to denatured DNA-cellulose were obtained. The predominant protein has a molecular weight of 20 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis and possesses many of the properties of the helix destabilizing proteins isolated from prokaryotic and eukaryotic sources. The protein facilitates denaturation of the synthetic copolymer poly[d(A-T).d(A-T)], depressing the melting temperature by nearly 40 degrees C. It also permits the renaturation of poly[d(A-T)].d(A-T)] in high salt concentration. Two other binding proteins have molecular weight of 25 000 and 23 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The protein with a molecular weight of 25 000 is probably the "M protein" previously isolated from Tetrahymena thermophila which has been shown to stimulate Tetrahymena DNA polymerase. These two proteins failed to show helix destabilizing, DNA dependent ATPase, or deoxyribonuclease activities. These three proteins are abundant in the cell with approximately 1.0 x 10(6) to 10.0 x 10(6) molecules of each protein monomer per cell. One molecule of each protein monomer binds to 7 to 10 nucleotides as detected by a nitrocellulose filter binding assay. Peptide mapping of the three proteins suggests that they are all distinct. We have also found that the binding proteins can interact with Tetrahymena DNA polymerase and some other proteins to form an enzyme complex, a putative replication complex.