Binding of enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene to polynucleotides

DNA covalent binding studies with enantiomers of trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (anti-BPDE) have been carried out by means of spectroscopic techniques (UV, CD, and fluorescence). Synthetic polynucleotides are employed to investigate binding differences between the G.C and A.T base pairs and to elucidate the bases for the stereoselective covalent binding of DNA toward anti-BPDE. The results indicate that of all the polynucleotides studied, only poly(dA-dT).poly(dA-dT) exhibits predominant intercalative covalent binding towards (+)-anti-BPDE and suffers the least covalent modification. Only minor intercalative covalent contributions are found in alternating polymer poly(dA-dC).poly(dG-dT). These observations parallel the DNA physical binding results of anti-BPDE and its hydrolysis products. They support the hypothesis that intercalative covalent adducts derive from intercalative physical binding while the external covalent adducts derive from external bimolecular associations. In contrast to the A.T polymers, the guanine containing polymers exhibit pronounced reduction in covalent modification by (-)-anti-BPDE. The intercalative covalent binding mode becomes relatively more important in the adducts formed by the (-) enantiomer as a consequence of decreased external guanine binding. These findings are consistent with the guanine specificity, stereoselective covalent binding at dG, the absence of stereoselectivity at dA for anti-BPDE, and the enhanced binding heterogeneity for the (-) enantiomer as found in the native DNA studies. The possible sequence and/or conformational dependence of such stereoselective covalent binding is indicated by the opposite pyrenyl CD sign exhibited by (+)-anti-BPDE bound to polynucleotides with pyrimidine on one strand and purine on another vs. that bound to polymers containing alternating purine-pyrimidine sequences.     

Facilitation of the conversion from B to Z conformation in poly(dG-dC) modified by anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide

The transition from B to Z conformation has been studied in poly(dG-dC) covalently modified with racemic anti- or syn-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a strong and a weak carcinogen, respectively. Circular dichroism was used to study the kinetics of the transition after a sudden increase of the ionic strength to 2.7 M NaCl. The results show that the rate of the B to Z transition of poly(dG-dC) in high NaCl concentration is considerably enhanced by bound anti-BPDE and diminished by bound syn-BPDE. The results may be interpreted such that at the binding site of anti-BPDE the base stacking is distorted and made looser, which facilitates the B to Z transition. The partly intercalative nature of the syn-BPDE complexes apparently is effective in reducing the rate of the transition. These properties of the two BPDEs may be relevant to explain their different carcinogenic potencies.     

Binding of Enantiomers of Trans-7, 8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo [a]pyrene to Polynucleotides

Abstract

DNA covalent binding studies with enantiomers of trans-7,8-dihydroxy- anti-9,10-epoxy- 7,8,9,10-tetrahydro-benzo [a] pyrene (anti-BPDE) have been carried out by means of spectroscopic techniques (UV, CD, and fluorescence). Synthetic polynucleotides are employed to investigate binding differences between the G · C and A · T base pairs and to elucidate the bases for the stereoselective covalent binding of DNA toward anti-BPDE. The results indicate that of all the polynucleotides studied, only poly(dA-dT) · poly(dA-dT) exhibits predominant intercalative covalent binding towards (+)-anti-BPDE and suffers the least covalent modification. Only minor intercalative covalent contributions are found in alternating polymer poly(dA-dC) · poly(dG-dT). These observations parallel the DNA physical binding results of anti-BPDE and its hydrolysis products. They support the hypothesis that intercalative covalent adducts derive from intercalative physical binding while the external covalent adducts derive from external bimolecular associations. In contrast to the A · T polymers, the guanine containing polymers exhibit pronounced reduction in covalent modification by (-)-anti-BPDE. The intercalative covalent binding mode becomes relatively more important in the adducts formed by the (-) enantiomer as a consequence of decreased external guanine binding. These findings are consistent with the guanine specificity, stereoselective covalent binding at dG, the absence of stereoselectivity at dA for anti-BPDE, and the enhanced binding heterogeneity for the (-) enantiomer as found in the native DNA studies. The possible sequence and/or conformational dependence of such stereoselective covalent binding is indicated by the opposite pyrenyl CD sign exhibited by (+)-anti-BPDE bound to polynucleotides with pyrimidine on one strand and purine on another vs. that bound to polymers containing alternating purine-pyrimidine sequences.     

Interaction of antimalarial drug quinacrine with nucleic acids of variable sequence studied by spectroscopic methods

The interaction of antimalarial drug quinacrine (QA) with polynucleotides is studied by UV-visible absorption, fluorescence and surface-enhanced Raman spectroscopy (SERS). The polynucleotides employed for such a study were calf thymus DNA, poly(A).poly(T), poly(A).poly(U), poly(C).poly(G) and poly(dG-dC).poly(dG-dC). Absorption and fluorescence spectra of QA complexes indicate that an interaction with the biomolecule is taking place, although different interaction mechanisms are probable depending on the sequence. The SERS spectra also reflect spectral changes which depend on the polymer sequence and that can be correlated to those observed by fluorescence, with the advantage of the detailed structural information provided by this vibrational technique. QA interacts with polynucleotides through its diprotonated form and by ring stacking. The strength of such interaction is extremely sequence dependent, thus suggesting different interaction mechanisms in each case. The SERS technique allows the simultaneous study of those polynucleotide moieties that are directly involved in the interaction thanks to the short-range character of the SERS spectroscopy. The interaction of QA with the above nucleic acids lead to a different change in the chain stability and flexibility which is further related to the different denaturation tendency of the polymer in the presence of the metal surface.     

Covalent binding of (+)- and (-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyr ene to B and Z DNAs

Circular dichroism (CD) as well as absorption spectral measurements reveals that poly(dG-m5dC).poly(dG-m5dC) suffers more extensive covalent modification by (+)-dihydroxy-anti-epoxybenzo[a]pyrene [(+)-anti-BPDE] than its unmethylated counterpart and that the covalently attached pyrenyl moiety exhibits stronger stacking interactions with the bases in the methylated polymer as suggested by the much larger pyrenyl spectral red shifts, most likely the consequence of intercalation. Stereoselective binding properties of these polymers are evidenced by the much reduced preference for the (-) enantiomer. Modifications due to (+)-anti-BPDE on the 50 microM hexaamminecobalt induced Z DNAs are much less pronounced and much less stereoselective, with the pyrenyl spectral characteristics being distinct from those of the B form. Salt titrations on the (+)-anti-BPDE modified poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) indicate much reduced cooperativity on the B to Z transition when compared to the unmodified counterparts. Evidence also suggests that covalent modification by anti-BPDE inhibits the B to Z conversion of base pairs in its immediate vicinity, presumably through intercalative stabilization of the B conformer at high salt. In contrast to stabilizing the B conformation for the proximal base pairs, covalent lesion by (+)-anti-BPDE appears to destabilize distal base pairs with the consequence of kinetic facilitation of B to Z transformation for these regions. Interesting differential effects on the reverse Z to B transforming abilities of these two enantiomers are observed with the covalent binding of the (-) isomer showing higher potency for inducing such conversion.     

Denaturation and renaturation studies of benzo[a]pyrene metabolite modified DNAs

Evidence from absorbance, fluorescence, and circular dichroism (CD) measurements strongly suggests that adduct conformations at the binding sites are grossly different before and after thermal denaturation of (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]py ren e [(+)-anti-BPDE] modified DNAs. This conclusion is reached through the following observations: (1) upon melting and cooling, the (+)-anti-BPDE-modified DNA exhibits pronounced hypochromic effects with concomitant spectral red shifts for the pyrenyl absorbance; (2) the pyrenyl CD spectrum reverses sign upon thermal denaturation-renaturation; (3) the fluorescence emission spectra resulting from excitations at 353 nm (10 nm to the red of hydrolyzed and unbound anti-BPDE) exhibit enhanced intensities and spectral red shifts for the thermally denatured and cooled adducts; and (4) in contrast to the absence of a shoulder prior to melting, the postmelt adducts exhibit a prominent 355-nm maximum (evidence of stacking interactions) in the excitation spectrum when 384-387-nm emission is monitored. Studies with synthetic polynucleotides further reveal that (+)-anti-BPDE-modified poly(dG).poly(dC) exhibits the greatest nonreversible renaturation at the binding sites, possibly as a consequence of pyrenyl self-stacking. This, coupled with the previous findings that this polymer suffers the most extensive (+)-anti-BPDE modification, appears to suggest that (dG)n . (dC)n regions may be responsible for such observed effects in native DNA.     

Interaction of Antimalarial Drug Quinacrine with Nucleic Acids of Variable Sequence Studied by Spectroscopic Methods

Abstract

The interaction of antimalarial drug quinacrine (QA) with polynucleotides is studied by UV- visible absorption, fluorescence and surface-enhanced Raman spectroscopy(SERS). The polynucleotides employed for such a study were calf thymus DNA, poly(A).poly(T), poly(A).poly(U), poly(C).poly(G) and poly(dG-dC).poly(dG-dC). Absorption and fluorescence spectra of QA complexes indicate that an interaction with the biomolecule is taking place, although different interaction mechanisms are probable depending on the sequence. The SERS spectra also reflect spectral changes which depend on the polymer sequence and that can be correlated to those observed by fluorescence, with the advantage of the detailed structural information provided by this vibrational technique. QA interacts with polynucleotides through its diprotonated form and by ring stacking. The strength of such interaction is extremely sequence dependent, thus suggesting different interaction mechanisms in each case. The SERS technique allows the simultaneous study of those polynucleotide moieties that are directly involved in the interaction thanks to the short-range character of the SERS spectroscopy. The interaction of QA with the above nucleic acids lead to a different change in the chain stability and flexibility which is further related to the different denaturation tendency of the polymer in the presence of the metal surface.     

Guanyl-C8-arylamination of DNA by the ultimate carcinogen of 4-nitroquinoline-1-oxide: a spectrophotometric titration

Native and denatured DNAs and polynucleotides were modified by 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide (4 NQO). The N-( deoxyguanosin -C8-yl)-4-aminoquinoline-1-oxide adduct, the so-called "dG III," was quantified on the DNA and on poly(dG-dC) in absorption spectroscopy, by using a spectral property of dG III, i.e., the variation of the absorption spectrum as a function of the pH. Using the "free-dG III" absorption reference spectra, a simple graphic determination of the percentage of dG III was established by recording the absorption spectra of the 4-acetoxyaminoquinoline-1-oxide-modified polymers. It was found that the dG III adduct accounts for about 30% of the total modification in the case of native modified DNA and poly(dG-dC) and for about 70% in the case of denatured modified DNA.     

Differential binding of the enantiomers of chloroquine and quinacrine to polynucleotides: Implications for stereoselective metabolism

Interaction of the antimalarial drugs quinacrine and chloroquine with DNA has been studied extensively in order to understand the origin of their biological activity. These studies have shown that they bind to DNA through an intercalative mode and show little sequence specificity. All previous experiments were carried out using the racemic form of these drugs. We have investigated the binding of the enantiomeric forms of quinacrine and chloroquine to synthetic polynucleotides poly (dA-dT) · poly(dA-dT) and poly (dG-dC) · poly(dG-dC), and found interesting differences in their binding parameters. Quinacrine enantiomers have a much higher binding affinity for the two polynucleotides compared to those of chloroquine. The negative enantiomers were found to have higher binding affinity than the positive ones. The binding constant for the binding of quinacrine (?) to poly(dG-dC) · poly(dG-dC) was found to be about 3 times that of quinacrine (+). The differences in these binding affinities were further confirmed by equilibrium dialysis of the complexes of the polynucleotides with the racemic form of the drugs, which resulted in the enrichment of the dialysate with the positive enantiomer. CD spectra of the enantiomers and their polynucleotide complexes are reported. Changes in the fluorescence properties of quinacrine in the presence of the two polynucleotides are also described. Biological implications of these findings are discussed. © 1993 John Wiley & Sons, Inc.     

Quinacrine: Spectroscopic properties and interactions with polynucleotides

The acridine dye quinacrine and its interactions with calf thymus DNA, poly(dA-dT) · poly (dA-dT), and poly (dG-dC) · poly(dG-dC) were studied by light absorption, linear dichroism, and fluorescence spectroscopy. The transition moments of quinacrine give rise to absorption bands polarized along the short axis (400–480-nm band), and the long axis (345-nm and 290-nm bands) of the molecule, respectively. Linear dichroism studies show that quinacrine intercalates into calf thymus DNA as well as into the polynucleotides, displaying fairly homogeneous binding to poly (dA-dT) · poly (dA-dT), but more than one type of intercalation site for calf thymus DNA and poly (dG-dC) · poly(dG-dC). Fluorescence spectroscopy shows that for free quinacrine the pK = 8.1 between the mono- and diprotonated states also remains unchanged in the excited state. Quinacrine bound to calf thymus DNA and polynucleotides exhibits light absorption typical for the intercalated diprotonated form. The fluorescence enhancement of quinacrine bound to poly (dA-dT) · poly(dA-dT) may be due to shielding from water interactions involving transient H-bond formation. The fluorescence quenching in poly(dG-dC) · poly(dG-dC) may be due to excited state electron transfer from guanine to quinacrine. © 1993 John Wiley & Sons, Inc.     

DNA sequence has an effect on the extent and kinds of alkylation of DNA by a potent carcinogen

A system has been developed to study the effects of base sequence (neighboring bases) upon the alkylation of guanine (G) and adenine (A) bases in DNA. The study was performed on the synthetic polydeoxyribonucleotides, poly(dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), as well as calf thymus DNA. Each polynucleotide was treated with N-[3H]methyl-N-nitrosourea (MNU), depurinated, and the freed alkylpurines separated by HPLC and quantitated by liquid scintillation counting. The amounts of 3-methylguanine (3-MG), 7-MG, and O6-MG relative to guanine, and 3-methyladenine (3-MA) and 1-MA plus 7-MA relative to adenine, and also the O6-MG/7-MG ratios were highly reproducible for a given polynucleotide. Significant differences were found in the amounts of each of the methylpurines formed when compared among the six synthetic polynucleotides and DNA. This evidence is interpreted as an effect upon alkylation which is ultimately dependent upon the base sequence. These findings may have significance in defining the specificity of chemical carcinogens in terms of the susceptability to modification of nucleotide sequences such as those found in certain oncogenes.     

Conformational effects of histones H1 on DNA structure. Comparative study between H1-1, H1(0), H5 and sperm holothuria phi 0

Interactions of mammalian histones, H1-1 and H1(0), phi 0 from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was phi 0 greater than H1-1 greater than H1(0). The binding of H1(0) to poly(dG-me5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1(0) with all polymers showed a strongly negative psi spectrum. Complexes of poly(dA-dT) and phi 0, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-like DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a psi spectrum. Complexes of poly(dG-me5dC) in the Z or B conformation with phi 0 showed spectral features characteristic of a mixture of a Z-like and a psi spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

Adduct formation between the carcinogen N-acetoxy-2-acetylaminofluorene and synthetic polydeoxyribonucleotides.

The chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF) was reacted with poly(dG-dC) - poly(dG-dC); poly dG - poly dC; poly(dA-dT) - poly (dA-dT); and poly dA - poly dT under a variety of conditions. Poly (dG-homo GC polymer and 10--20 more reactive the A + T polymers. Lowering the ionic strength increased the extent of reaction, while pH change (8.9 vs. 5.5) had only a small effect. If ionic strength was adjusted so that the two guanine-containing polymers showed equal thermal stability (as judged by Tm) then the alternating copolymer was 7 times as reactive as the homopolymer. In aggreement with previous investigators, the major product was found to be 8-(N-2-fluorenylacetamido) deoxyguanosine.     

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II)

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Conformational changes of poly(dG-dC) . poly(dG-dC) modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene.

Poly (dG-dC) . poly(dG-dC) was modified by the reaction with N-acetoxy-N-acetyl-2-aminofluorene. The conformations of poly(dG-dC) . poly(dG-dC) and of poly d(G-C)AAF were studied by circular dichroism under various experimental conditions. In 95% ethanol, the two polynucleotides adopt the A-form. In 3.9 M LiCl, the transition B-form-C-form is observed with poly(dG-dC) . poly (dG-dC) but not with poly d(G-C)AAF. In 1 mM phosphate buffer, poly d(G-C)AAF behaves as a mixture of B- and Z-form, the relative percentages depending upon the amounts of modified bases. The percentage of Z-form is decreased by addition of EDTA and is increased by addition of Mg++. Spermine favors the Z-form in modified and unmodified polynucleotides. No defect in the double helix of poly d(G-C)AAF is detected by SI endonuclease.     

Immunochemical approach for the characterization of DNA damages induced by chemical carcinogens

Mouse monoclonal antibody was elicited with 4-nitroquinoline 1-oxide (4NQO) modified poly(dG-dC).poly(dG-dC) and was characterized using enzyme-linked immunosorbent assay and radioimmunoassay. The antibody reacted specifically for 4NQO-poly(dG-dC).poly(dG-dC) but not for 4NQO modified DNA and synthetic polynucleotides such as poly(dG).poly(dC). The antibody crossreacted slightly with brominated or N-acetoxy-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) known to adopt Z-conformation. The antibody may recognize unique conformational change in poly(dG-dC).poly(dG-dC) modified by 4NQO. The antibody should be useful for the detection of conformational change in DNA induced by chemical carcinogens.     

Enzymatic methylation of chemically alkylated DNA and poly(dG-dC) X poly(dG-dC) in B and Z forms

The enzymatic methylation of chemically alkylated DNA and of poly(dG-dC) X poly(dG-dC) by beef brain DNA(cytosine-5-)-methyltransferase have been tested. The alkylation by dimethylsulfate, which yields mostly 7 methylguanine (m7G) and 3 methyladenine (m3A) do not affect the enzymatic methylation. The dimethylsulfate alkylated poly(dG-dC) X poly(dG-dC) converted into the Z-form in the presence of MgCl2, is just as well methylated as the native or the alkylated polynucleotide in the B-form. The alkylation of DNA or of poly(dG-dC) X poly(dG-dC) by methylnitrosourea yields, in addition to the above base modifications described for dimethylsulfate, methylphosphotriesters and O6-methylguanine. The enzymatic methylation of these substrates modified by methylnitrosourea is decreased. This decrease is proportional to the extent of the chemical alkylation of the substrate.     

Nucleotide sequence-dependent opening of double-stranded DNA at an electrically charged surface

It has been shown earlier that the DNA double helix is opened due to a prolonged contact of the DNA molecule with the surface of the mercury electrode. At neutral pH, the opening process is relatively slow (around 100 s), and it is limited to potentials close to -1.2 V (against SCE). The opening of the double helix has been explained by strains in the DNA molecule due to strong repulsion of the negatively charged phosphate residues from the electrode surface where the polynucleotide chain is anchored via hydrophobic bases. Interaction of the synthetic ds polynucleotides with alternating nucleotide sequences/poly(dA-dT).poly (dA-dT), poly (dA-dU).poly (dA-dU), poly (dG-dC).poly (dG-dC)/ and homopolymer pairs/poly (dA).poly (dT), poly (rA).poly (rU) and poly (dG).poly (dC)/ with the hanging mercury drop electrode has been studied. Changes in reducibility of the polynucleotides were exploited to indicate opening of the double helix. A marked difference in the behaviour was observed between polynucleotides with alternating nucleotide sequence and homopolymer pairs: opening of the double-helical structures of the former polynucleotides occurs at a very narrow potential range (less than 100 mV) (region U), while with the homopolymer pairs containing A X T or A X U pairs, the width of this region is comparable to that of natural DNA (greater than 200 mV). In contrast to natural DNA, the region U of homopolymer pairs is composed of two distinct phases. No region U was observed with poly (dG).poly (dC). In polynucleotides with alternating nucleotide sequence, the rate of opening of the double helix is strongly dependent on the electrode potential in region U, while in homopolymer pairs, this rate is less potential-dependent. It has been assumed that the difference in the behaviour between homopolymer pairs and polynucleotides with alternating nucleotide sequence is due to differences in absorbability of the two polynucleotide chains in the molecule of a homopolymer pair (resulting from different absorbability of purine and pyrimidine bases) in contrast to equal adsorbability of both chains in a polynucleotide molecule with alternating nucleotide sequence. It has been shown that the mercury electrode is a good model of biological surfaces (e.g. membranes), and that the nucleotide sequence-dependent opening (unwinding) of the DNA double helix at electrically charged surfaces may play an important role in many biological processes.     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.