Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.     

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards is preserved in intact cells.

Nitrogen mustard alkylating agents react with isolated DNA in a sequence selective manner, and the substituent attached to the drug reactive group can impose a distinct sequence preference. It is not clear however to what extent the observed DNA sequence preferences are preserved in intact cells. The highly reiterated sequence of human alpha DNA has been used to determine the sites of guanine-N7 alkylation following treatment of cells with three nitrogen mustards, mechlorethamine, uracil mustard and quinacrine mustard, known to react in isolated DNA with distinctly different sequence preferences. Alpha DNA from drug treated cells was extracted, purified, end-labeled, and a 296 base pair, singly end-labelled, fragment isolated. Following the quantitative conversion of alkylation sites to strand breaks the fragments were separated on DNA sequencing gels. Clear differences were observed between the alkylation patterns of the three compounds, and the selectivities were qualitatively similar to those predicted and observed in the same sequence alkylated in vitro. In particular the unique preferences of uracil and quinacrine mustards for 5'-PyGC-3' and 5'-GT/GPu-3' sequences, respectively, were preserved in intact cells suggesting that the pattern of sequence dependent reactivity is not grossly affected by the nuclear milieu.     

Mechanisms of DNA sequence selective alkylation of guanine-N7 positions by nitrogen mustards.

Quantitative determinations were carried out of the relative reaction rates of several nitrogen mustards at various guanine-N7 positions in DNA fragments of known sequence. The findings suggest structural hypotheses of the origins of the reaction selectivities. End-labeled DNA fragments were reacted with nitrogen mustards, and the guanine-N7 alkylation sites were analyzed by gel electrophoresis. Relative reaction intensities were determined by computer analysis of digitized densitometer scans. The differences in reaction intensities at different G's were in part attributable to the effects of nearest neighbor base pairs on the molecular electrostatic potential near the reaction site. Uracil and quinacrine mustards have specific sequence preferences for reaction that differ from other mustards. The nature of the specific sequence preferences were determined and hypotheses are proposed to explain their origin.     

DNA-directed alkylating ligands as potential antitumor agents: sequence specificity of alkylation by intercalating aniline mustards

The sequence preferences for alkylation of a series of novel parasubstituted aniline mustards linked to the DNA-intercalating chromophore 9-aminoacridine by an alkyl chain of variable length were studied by using procedures analogous to Maxam-Gilbert reactions. The compounds alkylate DNA at both guanine and adenine sites. For mustards linked to the acridine by a short alkyl chain through a para O- or S-link group, 5'-GT sequences are the most preferred sites at which N7-guanine alkylation occurs. For analogues with longer chain lengths, the preference of 5'-GT sequences diminishes in favor of N7-adenine alkylation at the complementary 5'-AC sequence. Magnesium ions are shown to selectively inhibit alkylation at the N7 of adenine (in the major groove) by these compounds but not the alkylation at the N3 of adenine (in the minor groove) by the antitumor antibiotic CC-1065. Effects of chromophore variation were also studied by using aniline mustards linked to quinazoline and sterically hindered tert-butyl-9-aminoacridine chromophores. The results demonstrate that in this series of DNA-directed mustards the noncovalent interactions of the carrier chromophores with DNA significantly modify the sequence selectivity of alkylation by the mustard. Relationships between the DNA alkylation patterns of these compounds and their biological activities are discussed.     

Use of [8-3H]guanine-labeled deoxyribonucleic acid to study alkylating agent reaction kinetics and stability.

Alkylation at the N7 position of guanine in DNA renders the C8-hydrogen acidic. This serves as the basis for an assay of guanine N7 alkylation using [8-3H]-guanine-labeled DNA. I modified the assay by preparing a high specific activity substrate in vitro and by replacing the distillation step with charcoal adsorption of substrate. Using the appearance of noncharcoal-adsorbable label as a measure of guanine-N7 alkylation I examined the reaction of DNA with dimethyl sulfate and mechlorethamine. The rate of reaction of dimethyl sulfate with the N7 position of guanine in DNA was constant over time, i.e., loss of label from DNA proceeded linearly with time. On the other hand, the rate of reaction of mechlorethamine with DNA increased with time, consistent with the initial formation of the reactive aziridinium ion. The assay can also be used to compare the reaction rates of various alkylating agents with DNA. Thus, the acridine mustards ICR-170 and quinacrine mustard were far more potent alkylating agents than mechlorethamine. Furthermore the assay may be used to determine the alkylating potency and stability of various alkylating agent preparations: while frozen solutions of acridine mustards in organic solvents retained alkylating activity for several months, different commercial preparations of quinacrine mustard had little or no alkylating activity.     

Specificity and kinetics of interstrand and intrastrand bifunctional alkylation by nitrogen mustards at a G-G-C sequence.

Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are potential sites for various bifunctional alkylations involving guanine N-7. To identify the DNA lesion(s) which may be responsible for these mutations, an end-labeled DNA duplex containing a frequent site of melphalan-induced mutation in the aprt gene was treated with melphalan, mechlorethamine or phosphoramide mustard. The sequence specificity and kinetics of formation of both interstrand and intrastrand crosslinks were determined. All mustards selectively formed two base-staggered interstrand crosslinks between the 5'G and the G opposite C in the 5'G-G-C sequence. Secondary alkylation was much slower for melphalan than for the other mustards and the resulting crosslink was more stable. Mechlorethamine and phosphoramide mustard induced intrastrand crosslinks between the two contiguous Gs in the G-G-C sequence in double-stranded DNA, but melphalan did not. Molecular dynamic simulations provided a structural explanation for this difference, in that the monofunctionally bound intermediates of mechlorethamine and phosphoramide mustard assumed thermodynamically stable conformations with the second arm in a position appropriate for intrastrand crosslink formation, while the corresponding melphalan monoadduct did not.     

Alteration in DNA cross-linking and sequence selectivity of a series of aziridinylbenzoquinones after enzymatic reduction by DT-diaphorase.

DT-diaphorase (DTD) mediated reduction of a series of 2,5-bis-substituted-3,6-diaziridinyl-1,4-benzoquinones was found to increase the level of DNA interstrand cross-linking (ISC) formed at neutral pH with an enhancement observed as the pH was decreased to 5.8. The analogues used were symmetrically alkyl-substituted carbamoyl ester analogues of AZQ (D1-D7), 3,6-diaziridinyl-1,4-benzoquinone (DZQ), the 2,5-dimethyl derivative (MeDZQ), and a 2,5-bis[(2-hydroxyethyl)amino] analogue (BZQ). At pH 5.8, the level of DNA ISC induced by enzymatic reduction was as follows: DZQ greater than MeDZQ much greater than D1 (methyl) greater than D3 (n-propyl) greater than D2 (AZQ; ethyl) greater than D5 (n-butyl) greater than D7 (sec-butyl) greater than D4 (isopropyl) D6 greater than (isobutyl). A similar trend was observed at pH 7.2. The level of DNA ISC induced by BZQ, which is not a substrate for DTD, was not increased by enzymatic reduction. Dicumarol, a known inhibitor of DTD, was capable of inhibiting the DNA ISC induced by these quinones upon enzymatic reduction. MeDZQ and DZQ reacted with guanines, as measured by Maxam and Gilbert sequencing, with a sequence selectivity similar to that of the nitrogen mustard class of antitumor agents. Enzymatic reduction of DZQ and MeDZQ by DTD was found to alter their sequence-selective alkylation. Reduced DZQ showed enhanced guanine alkylation in 5'-GC-3' sequences and new sites of adenine alkylation in 5'-(A/T)AA-3' sequences. Reduced MeDZQ only showed new sites of adenine alkylation at 5'-(A/T)AA-3' sequences but no enhancement of guanine alkylation. The new sites of adenine alkylation were found to be inhibited in the presence of magnesium and rapidly converted into apurinic sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA sequence-specific adenine alkylation by the novel antitumor drug tallimustine (FCE 24517), a benzoyl nitrogen mustard derivative of distamycin.

FCE 24517, a novel distamycin derivative possessing potent antitumor activity, is under initial clinical investigation in Europe. In spite of the presence of a benzoyl nitrogen mustard group this compound fails to alkylate the N7 position of guanine, the major site of alkylation by conventional nitrogen mustards. Characterisation of DNA-drug adducts revealed only a very low level of adenine adduct formation. Using a modified Maxam-Gilbert sequencing method the consensus sequence for FCE 24517-adenine adduct formation was found to be 5'-TTTTGA-3'. A single base modification in the hexamer completely abolishes the alkylation of adenine. Using a Taq polymerase stop assay alkylations were confirmed at the A present in the hexamer TTTTGA and, in addition, in one out of three TTTTAA sequences present in the plasmid utilized. The sequence specificity of alkylation by FCE 24517 is therefore the most striking yet observed for an alkylating agent of small molecular weight.     

Combinatorial identification of a novel consensus sequence for the covalent DNA-binding polyamide tallimustine

Many agents successfully used in cancer chemotherapy either directly or indirectly covalently modify DNA. Examples include cisplatin, which forms a covalent adduct with guanines, and doxorubicin, which traps a cleavage intermediate between topoisomerase II and torsionally strained DNA. In most cases, the efficacy of these drugs depends on the efficiency and specificity of their DNA binding, as well as the discrimination between normal and neoplastic cells in their handling of the drug-DNA adducts. While much is known about the chemistry of drug-DNA adducts, little is known regarding the overall specificity of their formation, especially in the context of a whole human genome, where potentially billions of binding sites are possible. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA-binding specificity of the semisynthetic covalent DNA-binding polyamide tallimustine, which contains a benzoic acid nitrogen mustard appended to the minor groove DNA-binding natural product distamycin A. After investigating over 134 million possible sequences, we found that the highest affinity tallimustine binding sites contained one of two consensus sequences, either the expected distamycin hexamer binding sites followed by a CG base pair (e.g., 5'-TTTTTTC-3' and 5'-AAATTTC-3') or the unexpected sequence 5'-TAGAAC-3'. Curiously, we found that tallimustine preferentially alkylated the N7 position of guanines located on the periphery of these consensus sequences. These findings suggested a cooperative binding model for tallimustine in which one molecule noncovalently resides in the DNA minor groove and locally perturbs the DNA structure, thereby facilitating alkylation by a second tallimustine of an exposed guanine on another side of the DNA.     

Quantum Molecular Modeling of the Interaction Between Guanine and Alkylating Agents - 2 - Nitrogen Mustard

Abstract

The alkylation mechanism of guanine by nitrogen mustard (HN2) was studied by using a supermolecular modeling at the ab initio 6–31G level. Our computations show that interaction of guanine with the aziridinium form of HN2 necessitates a transition state for the N7 alkylation route. The pathway of N7-guanine alkylation by nitrogen and sulfur mustards is discussed on the basis of the Molecular Electrostatic Potential and HOMO-LUMO properties of these molecules.     

Differences in sequence selectivity of DNA alkylation by isomeric intercalating aniline mustards

Two DNA-targeted mustard derivatives, N,N-bis(2-chloroethyl)-4-(5-[9-acridinylamino]-pentamido)aniline and 4-(9-[acridinylamino]butyl 4-(N,N-bis[2-chloroethyl]-aminobenzamide, which are isomeric compounds where the mustard is linked to the DNA-binding 9-aminoacridine moiety by either a -CONH- or a -NHCO- group, show significant differences in the sequence selectivity of their alkylation of DNA. The CONH isomer is a more efficient alxylating agent than the NHCO compound by an order of magnitude, consistent with the larger electron release of the CONH group to the aniline ring. However, the pattern of alkylation by the two compounds is also very different, with the CONH isomer preferring alkylation of guanines adjacent to 3'- or 5'-adenines and cytosines (for example those in sequences 5'-CGC, 5'-AGC, 5'-CGG and 5'-AGA) while the isomeric NHCO compound shows preference for guanines in runs of Gs. In addition, both isomers alkylate 3'-adenines in runs of adenines. Both compounds also show completely different patterns of alkylation to their untargeted mustard counterparts, since 4-MeCONH-aniline mustard alkylates all guanines and adenines in runs of adenines, while 4-Me2NCO-aniline mustard fails to alkylate DNA at all. These differences in alkylation patterns between the CONH- and its isomeric NHCO- compounds and their relationships between the alkylation patterns of the isomers and their biological activities are discussed.     

An application of 3D-QSAR to the analysis of the sequence specificity of DNA alkylation by uracil mustard.

The sequence specificity of DNA alkylation by uracil mustard was examined using a novel three-dimensional QSAR method known as HASL, or the hypothetical active site lattice. The structures of a variety of 4-mer sequences obtained from pBR322 and SV40 were related to their degree of guanine-N7 alkylation by uracil mustard. The resulting correlations were found to point to a significant contribution from bases on the 3' side of the target guanine nucleotide. The HASL models derived from the analysis of 52 guanine-containing 4-mer sequences were used to highlight those atomic features in the favored TGCC sequence that were found most important in determining specificity. It was found that the NH2-O systems present in the two GC base pairs on the 3' side of the target guanine were significantly correlated to the degree of alkylation by uracil mustard. This finding is consistent with a prealkylation binding event occurring between these sites along the major groove and the uracil mustard O2/O4 system.     

Mapping the binding site of aflatoxin B1 in DNA: systematic analysis of the reactivity of aflatoxin B1 with guanines in different DNA sequences

The mutagenic and carcinogenic chemical aflatoxin B1 (AFB1) reacts almost exclusively at the N(7)-position of guanine following activation to its reactive form, the 8,9-epoxide (AFB1 oxide). In general N(7)-guanine adducts yield DNA strand breaks when heated in base, a property that serves as the basis for the Maxam-Gilbert DNA sequencing reaction specific for guanine. Using DNA sequencing methods, other workers have shown that AFB1 oxide gives strand breaks at positions of guanines; however, the guanine bands varied in intensity. This phenomenon has been used to infer that AFB1 oxide prefers to react with guanines in some sequence contexts more than in others and has been referred to as "sequence specificity of binding". Herein, data on the reaction of AFB1 oxide with several synthetic DNA polymers with different sequences are presented, and (following hydrolysis) adduct levels are determined by high-pressure liquid chromatography. These results reveal that for AFB1 oxide (1) the N(7)-guanine adduct is the major adduct found in all of the DNA polymers, (2) adduct levels vary in different sequences, and, thus, sequence specificity is also observed by this more direct method, and (3) the intensity of bands in DNA sequencing gels is likely to reflect adduct levels formed at the N(7)-position of guanine. Knowing this, a reinvestigation of the reactivity of guanines in different DNA sequences using DNA sequencing methods was undertaken. The reactivities of 190 guanines were determined quantitatively and considered in a pentanucleotide context, 5'-WXGYZ-3', where the central, underlined G represents the reactive guanine and W, X, Y, and Z can be any of the nucleotide bases. Methods are developed to determine that the X (5'-side) base and the Y (3'-side) base are most influential in determining guanine reactivity. The influence of the bases in the 5'-position (X) is 5'-G (1.0) greater than C (0.8) greater than A (0.3) greater than T (0.2), while the influence of the bases in the 3'-position (Y) is 3'-G (1.0) greater than T (0.8) greater than C (0.4) greater than A (0.3). These rules in conjunction with molecular modeling studies (to be published elsewhere) were used to assess the binding sites that might be utilized by AFB1 oxide in its reaction with DNA.     

The sequence specificity of alkylation for a series of benzoic acid mustard and imidazole-containing distamycin analogues: the importance of local sequence conformation.

The covalent sequence specificity of a series of nitrogen mustard and imidazole-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two or three imidazole units. Examination of the alkylation specificity revealed that BAM produced guanine-N7 lesions in a pattern similar to conventional nitrogen mustards. The monoimidazole-BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM, but at a 100-fold lower dose. The diimidazole and triimidazole conjugates did not produce detectable guanine-N7 alkylation but only alkylated at selected sites in the minor groove. Unexpectedly, the alkylation specificity at equivalent doses was nearly identical to that found for the previously reported pyrrole-BAM conjugates. The consensus sequence, 5'-TTTTGPuwas strongly alkylated by the triimidazole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies were carried out to examine the non-covalent DNA binding interactions. These studies revealed that the tripyrrole- BAM conjugate bound non-covalently to the same AT-rich sites as distamycin. In contrast, whereas the Im3lexitropsin bound non-covalently to GC-rich sequences, the triimidazole-BAM conjugate did not detectably footprint to either GC- or AT-rich regions at equivalent doses. The results indicate that the alkylation event is not solely dictated by the non-covalent binding and might be influenced by a unique sequence dependent conformational feature of the consensus sequence 5'-TTTTGPu.     

Sequence-specific spin labeling of DNA

Several DNA fragments deriving from plasmid pBR322 were used to determine the modification sites caused by the reaction with alkylating spin-labeling probes. At a high spin-label concentration, all guanines became alkylated, causing the cleavage of the phosphodiester bonds upon the treatment with piperidine. The lengths of the breakage products of 5'-end labeled DNA treated with spin labels were compared with the length of DNA scission products generated by Maxam-Gilbert procedure for DNA sequence analysis. The distribution of the guanine modifications is dependent on the amount of the reagent used for the alkylation and the ionic conditions of the reaction. The frequency of alkylation by spin labels was greatly enhanced within continuous runs of guanines in DNA. The stabilization of the DNA structure by magnesium or spermine directs the spin-label binding specifically to the most exposed region of DNA fragment containing GGTGG sequence. The sequence-dependent interaction of spin labels with DNA enables the development of the method for the selective spin labeling of DNA molecule.     

Formation and structural determinants of multi-stranded guanine-rich DNA complexes

We have investigated the complexes formed by oligonucleotides with the general sequence d(T15,Gn), where n = 4-15. Two distinct classes of structures are formed, namely, the four-stranded tetraplex and frayed wires. Frayed wires differ from four-stranded tetraplexes in both strand association stoichiometry and the ability of dimethyl sulfate to methylate the N7 position of guanine. Thus, it appears that these two guanine-rich multistranded assemblies are stabilised by different guanine-guanine interactions. The number of contiguous guanine residues determines which of the complexes is favoured. Based on the stoichiometry of the associated species and the accessibility of the N7 position of guanine to methylation we have found that oligonucleotides with smaller number of contiguous guanines; n = 5-8, form primarily four-stranded tetraplex. Oligonucleotides with larger numbers of contiguous guanines adapt primarily the frayed wire structure. The stability of the complexes formed by this series of oligonucleotides is determined by the number and arrangement of the guanines within the sequences. We propose that the formation of the two types of complex proceed by a parallel reaction pathways that may share common intermediates.     

Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen mustard analogues in an SV40-based shuttle plasmid.

It was previously shown that the predominant mutations induced by melphalan (L-phenylalanine mustard) in the supF gene of shuttle plasmid pZ189 during replication in human cells are A.T----T.A transversions. In order to determine whether adenine adducts were formed at sequence positions corresponding to these mutations, melphalan-induced thermolabile adducts were mapped in the supF gene by selective depurination followed by strand cleavage in alkali. All A.T base pairs which were frequent sites for melphalan-induced A.T----T.A transversions were also prominent sites for formation of thermolabile adenine adducts. Although no mutations were detected at some prominent adduct sites, there was a significant correlation between adduct sites and mutation sites. While runs of two or more adenines were particularly prominent adduct sites, comparison of results obtained with 3'- and 5'-end-labeled DNA gave no evidence for intrastrand cross-links between adjacent adenines. Chlorambucil, another aromatic nitrogen mustard, showed sequence specificities for both mutagenesis and adenine adduct formation nearly identical to those seen with melphalan. The nonaromatic analogues mechlorethamine and phosphoramide mustard were much less efficient in inducing thermolabile adenine adducts, and mechlorethamine induced significantly fewer transversions at A.T base pairs than chlorambucil or melphalan. Formation of thermolabile adenine adducts by the aromatic nitrogen mustards was markedly reduced by blockage of the minor groove with distamycin, or by prior heat denaturation of the DNA. These results suggest that alkylation occurs primarily at the N-3 rather than N-7 position of adenine, probably as a consequence of the affinity of the aromatic rings of melphalan and chlorambucil for the minor groove.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations induced by some DNA minor groove binding alkylators in AS52 Chinese hamster cells

Nitrogen mustards are commonly used in cancer chemotherapy. They interact with DNA at electronegative sites, primarily forming N7 guanine mono-adducts and interstrand cross-links. Targeting nitrogen mustards to DNA by attachment of a DNA minor groove binding carrier such as the bisbenzimidazoles Hoechst 33258 (pibenzimol) or Hoechst 33342 (HOE) makes it possible to direct DNA alkylation to more specific stretches of DNA. We have performed a detailed molecular analysis of 6-thioguanine resistant clones arising in Chinese hamster AS52 cells after treatment with HOE, in comparison with a mono- and bifunctional pair of bisbenzimidazole-targeted nitrogen mustards (MGBs). HOE showed no significant ability to induce 6-thioguanine resistant mutants, possibly because drug-treated cells are highly susceptible to apoptosis within very short times. Neither of the MGBs caused the rapid cell death seen with the bisbenzimidazole. However, both MGBs were weaker mutagens than previously found for undirected mustards in the same system, an effect that we suggest could relate to greater structure-directed binding to less mutable DNA sites in the minor groove. Additionally, the nature of some of the mutants suggested there may be a small component of topo I and/or II-mediated events in the mutagenicity of the MGBs. Both MGBs showed high activity in causing deletion mutations, which may be due to errors in attempted repair of the complex lesions formed by minor groove targeted alkylators.     

Kinetics of DNA alkylation, depurination and hydrolysis of anti diol epoxide of benzo(a)pyrene and the effect of cadmium on DNA alkylation

Anti benzo[a]pyrene diol epoxide (BPDE) alkylates guanines of DNA at N7 in the major groove and at the exocyclic amino group in the minor groove. In this report we investigated the rates of BPDE hydrolysis, DNA alkylation and subsequent depurination of BPDE-adducted pBR322 DNA fragment using polyacrylamide gel electrophoresis. Preincubation studies showed that it hydrolyzed completely in triethanolamine buffer in <2 min. The depurination kinetics showed that a fraction of the N7 alkylated guanine depurinated rapidly; however a significant amount of N7 guanine alkylation remained stable to spontaneous depurination over a 4-h period. Similar results were obtained for the hydrolysis and alkylation rates of syn isomer but it required nearly 500 times more concentration to induce similar levels of N7 guanine alkylation. Cadmium ion strongly inhibited the N7 guanine alkylation of both isomers. But the minor groove alkylation was not affected as demonstrated by postlabeling assay which confirmed the presence of heat-and cadmium-stable minor groove adducts in BPDE-treated calf thymus DNA. Based on these and our earlier findings, we propose a mechanism for the synergistic effect of cadmium in chemically induced carcinogenesis.     

DNA sequence recognition by the antitumor drug ditercalinium

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.