Comparison between poly(dG-dC).poly(dG-dC) and DNA modified by cis-diamminedichloroplatinum (II): immunological and spectroscopic studies

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG).poly(dC) is larger than to poly (dG-dC).poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT). In the competition between poly(dG-dC).poly (dG-dC) (B conformation) and poly(dG-br5dC).poly(dG-br5dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)n.(GC)n sequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diamminedichloroplatinum (II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m5dC) in the Z conformation. When they bind to poly(dG-m5dC) in the B conformation, the conformations of poly(dG-m5dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.     

A pulse-radiolysis study of the reaction of hydrated electrons with N'-formylkynurenine and related compounds: electron-transfer reactions with nucleic acid components.

The reactivity of N'-formylkynurenine (FK) derivatives towards eaq has been investigated. The reduced transient species have been characterized (lambda max approximately 340, 440 nm, epsilon lambda max approximately 3000-1000 M-1 cm-1, pKa approximately 7.8). Owing to the strong FK electron affinity, electron-transfer reactions occur from purine (except guanine) and pyrimidine electron adducts to FK (k approximately 2-7 x 10(9) M-1 s-1). As some FK derivatives bind to DNA (or polynucleotides) the protective effect of complexation on FK-DNA (or polynucleotides) adduct formation has been investigated.     

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.     

Conformations and selective photodissociation of heterogeneous benzo(a)pyrene diol epoxide enantiomer-DNA adducts

The covalent binding of the tumorigenic (+) enantiomer and the nontumorigenic (-) enantiomer of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,19-tetrahydrobenzo(a)pyrene (BPDE) to double-stranded native DNA gives rise to heterogeneous adducts, especially in the case of (-)-BPDE. The covalent (+)-BPDE-DNA adducts are predominantly of the external site II type, while the (-)-BPDE-DNA adducts are predominantly of the quasi-intercalative, site I type (65%), with 35% of site II adducts. The site I adducts can be selectively photodissociated with near-ultraviolet light (quantum yields in the range 0.0003-0.005); the external site II adducts (photodissociation quantum yield 3 X 10(-5) are 10-100-times more stable. The photolability of covalent (-)-BPDE-DNA adducts accounts for the discrepancies in the linear dichroism properties of these complexes reported previously. Fluorescence quenching data, previously utilized to assess the degree of solvent exposure of the pyrenyl residues in covalent adducts, were in some cases significantly influenced by the presence of highly fluorescent tetraol dissociation products. After correcting for this effect, it is shown that the fluorescence of the external site II (+)-BPDE-DNA adducts is sensitive to acrylamide, while the fluorescence of the dominant site I (-)-BPDE-DNA adducts is not affected by this fluorescence quencher, as expected for adducts with considerable carcinogen-base stacking interactions.     

Repair of DNA lesions: mechanisms and relative repair efficiencies

DNA is frequently damaged by endogenous agents inside the cells. Some exogenous agents such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and may thus contribute to the 'background' DNA damage in humans. DNA lesions are normally removed by various repair mechanisms. The major repair mechanisms for various DNA lesions are summarized. In contrast to the extensively studied repair mechanisms, much less is known about the relative repair efficiencies of various DNA lesions. Since DNA repair is a crucial defense against carcinogenesis, it may constitute an important factor affecting the carcinogenicity of DNA damaging agents. We have adopted a human cell-free system for measuring relative DNA repair efficiencies based on the concept of repair competition between acetylaminofluorene adducts and other DNA lesions of interest. Using this in vitro system, we determined the relative repair efficiencies of PAH adducts induced by: anti-(+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), anti-(+/-)-benz[a]anthracene-trans-3,4-dihydrodiol-1,2-epoxide (BADE-I), anti-(+/-)-benz[a]anthracene-trans-8,9-dihydrodiol-10, 11-epoxide (BADE-II), anti-(+/-)-benzo[b]fluoranthene-trans-9, 10-dihydrodiol-11,12-epoxide (BFDE), anti-(+/-)-chrysene-trans-1, 2-dihydrodiol-3,4-epoxide (CDE), and anti-(+/-)-dibenzo[a, l]pyrene-trans-11,12-dihydrodiol-13,14-epoxide (DBPDE). While damage by BPDE, DBPDE, CDE, and BFDE were repaired by nucleotide excision repair as efficiently as AAF adducts, the repair of BADE-I and BADE-II adducts were significantly slower in human cell extracts. Damage by DBPDE at 3 microM in vitro yielded approximately 5-fold higher DNA adducts than BPDE as determined by quantitative PCR. This potent DNA reactivity may account in part for the potent carcinogenicity of dibenzo[a,l]pyrene. The correlation of these results to the carcinogenic properties of the PAH compounds is discussed. Furthermore, we show that NER plays a role in AP site repair in vivo in the eukaryotic model organism yeast.     

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 arsenic trioxide As2O3 with DNA and RNA

Arsenic salts have been used for centuries to treat a variety of medical conditions ranging from infectious disease to cancer. More recently, trivalent arsenic trioxide was found to exhibit high antitumor activity towards hematological malignancies. Even though much is known about antitumor activity and DNA damage by As2O3, there has been no report on the interaction of arsenic trioxide with isolated DNA or RNA. Therefore, it was of interest to examine the interaction of As2O3 with DNA and RNA in aqueous solution at physiological pH. FTIR and UV-visible difference spectroscopic methods were used to characterize the nature of drug-DNA and drug-RNA interactions and to determine the As binding site, the binding constant, the sequence selectivity, the helix stability, and the biopolymer secondary structure in the As2O3-polynucleotide complexes in vitro. The FTIR spectroscopic studies were conducted with As2O3-polynucleotide (phosphate) ratios of 1/40, 1/20, 1/10, and 1/5, with a final DNA (P) or RNA (P) concentration of 6.25 mmol/l. Spectroscopic results showed As2O3 binds to DNA and RNA at G-C, A-T, and A-U bases, and no interaction with the backbone PO2 group. As2O3-DNA and -RNA adducts showed one type of binding with overall binding constant of K(As2O3-DNA) = 1.24 x 10(5) M(-1) and K(As2O3-RNA) = 2.60 x 10(5) M(-1). The As2O3-polynucleotide complexation is associated with a partial biopolymer aggregation and no major alterations of B-DNA or A-RNA structure.     

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).     

Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption

We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism. Upon complex formation the absorption spectra of both the protein and the polynucleotides change. The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT)8. All gp5-polynucleotide complexes give rise to intense linear dichroism spectra. These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned. The spectra can be explained by polynucleotide structures, which are the same in all complexes. The base orientations are characterized by a substantial inclination and propellor twist. The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited. The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.     

The formation of acetylaminofluorene adducts in poly(dC-dG) and poly(dA-dT) on reaction with N-acetoxy-2-acetylaminofluorene and the effect of such modification upon the polymers as templates for DNA polymerases

N-Acetoxy-2-acetylaminofluorene (AcO-AAF) reacts with the alternating DNA-like polynucleotides poly(dC-dG) and poly(dA-dT) in vitro to give adducts of the guanine and adenine bases similar to those reported to be formed in DNA. A previously unobserved guanine adduct was detected in the poly(dC-dG). Using a double-labelled [U-14C-dG, 8-3H-G]-poly(dC-dG) we show that this adduct does not involve the 7- or 8-positions of the guanine. Similarly a thymine adduct of unknown structure was observed in poly(dA-dT). Modification of the polymers with AcO-AAF inhibits their capacity to act as templates for Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha although the binding of the polymerases to the polynucleotides is unaffected. Such modification also leads to an increase in the levels of non-complementary nucleotides incorporated into newly synthesised DNA.     

Binding of pyrene to DNA, base sequence specificity and its implication.

Solubilization as well as spectral studies of pyrene in natural DNA and synthetic deoxypolynucleotide solutions at neutral pH reveal at least two binding modes. Sites I are predominant in native DNA and in poly(dA-dT): poly(dA-dT) whereas sites II are found with denatured DNA and other polynucleotides such as poly(dA):poly(dT) and three different types of guanine containing copolymers which solubilize pyrene to a lesser extent. Spectral comparison with the covalent adducts of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo(a)pyrene (anti-BPDE) and the physical complexes of its tetraols lead to the suggestion of a base sequence specific binding model for this carcinogenic metabolite to account for the puzzling fact that although its physical binding is predominantly intercalative, the covalent adducts appear not to be intercalated. It is speculated that in neutral solutions, intercalation may have little, if any, to do with the chemical lesion of this metabolite to the guanine base of the DNA and may, on the contrary, provide an efficient pathway for detoxification.     

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.     

Quantification of the DNA cleavage and packaging proteins U(L)15 and U(L)28 in A and B capsids of herpes simplex virus type 1

The proteins produced by the herpes simplex virus type 1 (HSV-1) genes U(L)15 and U(L)28 are believed to form part of the terminase enzyme, a protein complex essential for the cleavage of newly synthesized, concatameric herpesvirus DNA and the packaging of the resultant genome lengths into preformed capsids. This work describes the purification of recombinant forms of pU(L)15 and pU(L)28, which allowed the calculation of the average number of copies of each protein in A and B capsids and in capsids lacking the putative portal encoded by U(L)6. On average, 1.0 (+/-0.29 [standard deviation]) copies of pU(L)15 and 2.4 (+/-0.97) copies of pU(L)28 were present in B capsids, 1.2 (+/-0.72) copies of pU(L)15 and 1.5 (+/-0.86) copies of pU(L)28 were found in mutant capsids lacking the putative portal protein pU(L)6, and approximately 12.0 (+/-5.63) copies of pU(L)15 and 0.6 (+/-0.32) copies of pU(L)28 were present in each A capsid. These results suggest that the packaging machine is partly comprised of approximately 12 copies of pU(L)15, as found in A capsids, with wild-type B and mutant U(L)6(-) capsids containing an incomplete complement of cleavage and packaging proteins. These results are consistent with observations that B capsids form by default in the absence of packaging machinery in vitro and in vivo. In contrast, A capsids may be the result of initiated but aborted attempts at DNA packaging, resulting in the retention of at least part of the DNA packaging machinery.     

Carcinogenic purine N-oxide ester modifies covalently all common bases in polynucleotides

The carcinogen 1-methyl-3-hydroxyxanthine after esterification binds covalently to polynucleotides, RNA and DNA. All four ribopolynucleotides and poly(dT) are targets. Depending on reaction conditions, covalent binding is greatest to poly(A) followed by poly(U), poly(dT), poly(G), poly(C), RNA and DNA. Maximal covalent modification of DNA is one moiety per 360 nucleotides. All modified polynucleotides, RNA and DNA, except poly guanylic acid have been enzymatically digested and the major adducts characterized as nucleosides.     

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.     

Stereoselective covalent binding of anti-benzo(a)pyrene diol epoxide to DNA conformation of enantiomer adducts

The conformation of adducts derived from the reactions and covalent binding of the (+) and (-) enantiomers of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) with double-stranded calf thymus DNA in vitro were investigated utilizing the electric linear dichroism technique. The linear dichroism and absorption spectra of the covalent DNA complexes are interpreted in terms of a superposition of two types of binding sites. One of these conformations (site I) is a complex in which the plane of the pyrene residue is close to parallel (within 30 degrees) to the planes of the DNA bases (quasi-intercalation), while the other (site II) is an external binding site; this latter type of adduct is attributed to the covalent binding of anti-BaPDE to the exocyclic amino group of deoxyguanine (N2-dG), while site I adducts are attributed to the O6-deoxyguanine and N6-deoxyadenine adducts identified in the product analysis of P. Brookes and M.R. Osborne (Carcinogenesis (1982) 3, 1223-1226). Site II adducts are dominant (approximately 90% in the covalent complexes derived from the (+) enantiomer), but account for only 50 +/- 5% of the adducts in the case of the (-)-enantiomer. The orientation of site II complexes is different by 20 +/- 10 degrees in the adducts derived from the binding of the (+) and the (-) enantiomers to DNA, the long axis of the pyrene chromophore being oriented more parallel to the axis of the DNA helix in the case of the (+) enantiomer. These findings support the proposals by Brookes and Osborne that the difference in spatial orientation of the N2-dG adducts of (-)-anti-BaPDE together with their lower abundance may account for the lower biological activity of the (-) enantiomer. The external site II adducts, rather than site I adducts, appear to be correlated with the biological activity of these compounds.     

Salt-dependent binding of Escherichia coli initiation factor 3 to nucleic acids determined by sedimentation partition chromatography

The binding of 14CH3- initiation factor 3 (IF3) to polynucleotides is strongly dependent upon the concentration of added salt. The observed association constant, Kobs, increases by ca. a factor of 10(2) when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to all nucleic acids examined. This salt-dependent binding suggests that at physiological salt concentrations the formation of an IF3-polynucleotide complex is primarily driven by the release of cations from the nucleic acid, although anion effects are involved also. For single-stranded nucleic acids, nonelectrostatic interactions may contribute a factor of 10(2) to the value of Kobs, although accurate assessment of these interactions is complicated by anion effects. The binding of 14CH3-IF3 to the double helix, poly(A).poly(U), appears to be exclusively electrostatic. 14CH3-IF3 forms a maximum of 8 +/- 2 ion pairs with most single-stranded polynucleotides. The value of Kobs increases from ca. 10(3) to 10(5) M-1 when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to poly(A), poly(C), poly(U), and poly(A).poly(U). At physiological salt concentrations, IF3 shows no preference for any of these bases or for single or double-stranded structures. However, 14CH3-IF3 binds ca. 60 times greater to poly(A,G), at al NaCl concentrations examined, than to the other nucleic acids, indicating that IF3 has some preference for guanine-containing polynucleotides. The presence of 10 mM Mg2+ tends to reduce the value of Kobs at any given NaCl concentration, but to a smaller degree than predicted by simply a competition between Mg2+ and IF3 for the nucleic acid lattice.     

Covalent binding studies on the 14C-labeled antitumor compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone. Involvement of semiquinone radical in binding to DNA, and binding to proteins and bacterial macromolecules in situ.

2,5-Bis(1-aziridinyl)-1,4-benzoquinone (BABQ) is a compound from which several antitumour drugs are derived, such as Trenimone, Carboquone and Diaziquone (AZQ). The mechanism of DNA binding of BABQ was studied using 14C-labeled BABQ and is in agreement with reduction of the quinone moiety and protonation of the aziridine ring, followed by ring opening and alkylation. The one-electron reduced (semiquinone) form of BABQ alkylates DNA more efficiently than two-electron reduced or non reduced BABQ. Covalent binding to polynucleotides did not unambiguously reveal preference for binding to specific DNA bases. Attempts to elucidate further the molecular structure of DNA adducts by isolation of modified nucleosides from enzymatic digests of reacted DNA failed because of instability of the DNA adducts. The mechanism of covalent binding to protein (bovine serum albumin, BSA) appeared to be completely different from that of covalent binding to DNA. Binding of BABQ to BSA was not enhanced by reduction of the compound and was pH dependent in a way that is opposite to that of DNA alkylation. Glutathione inhibits binding of BABQ to BSA and forms adducts with BABQ in a similar pH dependence as the protein binding. The aziridine group therefore does not seem to be involved in the alkylation of BSA. Incubation of intact E. coli cells, which endogenously reduce BABQ, resulted in binding to both DNA and RNA, but also appreciable protein binding was observed.     

Electron microscopic measurement of chain flexibility of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II).

The antitumor drug cis-diamminedichloroplatinum (II) (cis-Pt) forms bidentate adducts with guanine residues of poly(dG-dC).poly(dG-dC). The secondary structure of the polymer is altered. In this work, high resolution pictures of naked molecules, obtained by dark field electron microscopy reveal DNA chain distortions with radii as small as 30 A. The extent of distortion increases with the drug/nucleotide ratio (rb). These alterations of the secondary structure are responsible for the apparent shortening of the molecules. Measurements of the persistence lengths of the polymer as well as the end-to-end distances of elementary segments of various lengths, are obtained from digitized electron micrographs. The measurements are used to monitor and quantify the observed modifications of polymer structure upon cis-Pt binding at various rb or incubation times. Poly(dG-m5dC).poly(dG-m5dC) in the B and Z forms have different persistence lengths. In the B form, this polymer is more altered by cis-Pt than in the Z one.     

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.