Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-t rimethylammoniumethyl-amine)phenyl]porphy rin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine )phenyl]porphyrin (TthetaF4TAP) has been synthesized. The binding interactions of TthetaF4TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TthetaF4TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TthetaF4TAP binds to calf thymus DNA, [poly(dA-dT)]2, and [poly(dG-dC)]2 in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TthetaF4TAP/CT DNA resembled that of TthetaF4TAP/[poly(dA-dT)]2, suggesting that TthetaF4TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TthetaF4TAP binds preferentially to [poly(dA-dT)]2 over [poly (dG-dC)]2. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TthetaF4TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TthetaF4TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation.     

GC base sequence recognition by oligo(imidazolecarboxamide) and C-terminus-modified analogues of distamycin deduced from circular dichroism, proton nuclear magnetic resonance, and methidiumpropylethylenediaminetetraacetate-iron(II) footprinting studies

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of pre-existing methylation on the de novo activity of eukaryotic DNA methyltransferase

Aberrant de novo methylation of CpG island DNA sequences has been observed in cultured cell lines or upon malignant transformation, but the mechanisms underlying this phenomenon are poorly understood. Using eukaryotic DNA (cytosine-5)-methyltransferase (of both human and murine origin), we have studied the in vitro methylation pattern of three CpG islands. Such sequences are intrinsically poor substrates of the enzyme, yet are efficiently methylated when a small amount of 5-methylcytosine is randomly introduced by the M.SssI prokaryotic DNA (cytosine-5)-methyltransferase prior to in vitro methylation by the eukaryotic enzyme. A stimulation was also found with several other double-stranded DNA substrates, either natural or of synthetic origin, such as poly(dG-dC).poly(dG-dC). An A + T-rich plasmid, pHb beta 1S, showed an initial stimulation, followed by a severe inhibition of the activity of DNA (cytosine-5)-methyltransferase. Methylation of poly(dI-dC).poly(dI-dC) was instead inhibited by pre-existing 5-methylcytosines. The extent of stimulation observed with poly(dG-dC).poly(dG-dC) depends on both the number and the distribution of the 5-methylcytosine residues, which probably must not be too closely spaced for the stimulatory effect to be exerted. The activity of the M.SssI prokaryotic DNA methyltransferase was not stimulated, but was inhibited by pre-methylation on either poly(dG-dC).poly(dG-dC) or poly(dI-dC).poly(dI-dC). The prokaryotic and eukaryotic DNA methyltransferases also differed in sensitivity to poly(dG-m5dC).poly(dG-m5dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.     

Structural characterization of the 1:1 adduct formed between the antitumor antibiotic hedamycin and the oligonucleotide duplex d(CACGTG)2 by 2D NMR spectroscopy

2D NMR spectroscopic methods have been used to determine the structure of the adduct formed between the antitumor antibiotic hedamycin and the oligodeoxyribonucleotide duplex d(CACGTG)2. Evidence for both intercalation and alkylation in the adduct was observed, and a model for the binding interaction was constructed based on intermolecular NOEs and distance-restrained molecular dynamics. In our computationally refined model, the anthrapyrantrione chromophore of hedamycin is intercalated between the 5'-CG-3' bases with the two aminosugar groups placed in the minor groove and the six carbon bisepoxide side chain located in the major groove. The anglosamine sugar attached at C8 is oriented in the 3' direction relative to the intercalation site, while the N,N-dimethylvancosamine attached at C10 is oriented to the 5' side, with each aminosugar wedged between a guanine exocyclic amino group and one of the groove walls. The terminal epoxide carbon C18 is covalently bound to the N7 atom of the central guanine, as evidenced by lability of the C8 hydrogen of this purine upon reaction with hedamycin. Our binding model places the C10-attached N,N-dimethylvancosamine of hedamycin in van der Waals contact with the alkylated strand. A strong NOE contact verifies the close proximity of the terminal methyl group (C19) of the bisepoxide side chain to the methyl group of the thymine on the 3' side of the alkylated guanine. This, in conjunction with other data, suggests hydrophobic interactions between the bisepoxide chain and the floor of the major groove may contribute to sequence recognition. Furthermore, it is proposed that the 5'-CGT sequence selectivity of hedamycin arises, in part, from complementarity in shape between the chromophore substituents and the major and minor groove at the binding site.     

DNA receptor sites for the intercalation of nogalamycin

The intercalation of the planar chromophoric moiety of nogalamycin between two base pairs of duplex DNA has been evidenced by means of low-dichroism measurements. The possible presence of specific binding sites for mogalamycin on DNA has been suggested by studies on the denaturation and renaturation of DNA complexed with nogalamycin. A clear evidence was obtained by investigating the interaction of nogalamycin with polydeoxyribonucleotides containing known, regularly repeating sequences, used as model compounds. The results obtained with these polymers and the DNA suggest that the segment containing both purine (A,G) anf pyrimidine (T,C) bases in alternate sequences is the preferential receptor site on the DNA. A decreasing affinity is exhibited by poly d(A--T)-poly d(A--T), poly d(G--C)-poly d(G--C) and poly dG-poly dC segments, in the order. The poly dA-poly dT sequence appears to be closed to the interaction of nogalamycin.     

Change in capsulorhexis size with four foldable loop-haptic lenses over 6 months

The interaction of a series of potent leishmanicidal aromatic diamidines resembling pentamidine, was studied with Leishmania infantum DNA and polynucleotides. The diamidines viz., CGP040215A, CGP033829A and CGP039937A, interacted with leishmania DNA as well as with the polynucleotides poly(dA)-poly(dT), poly(dA-dT) and poly(dG-dC). The thermodynamic analysis to determine the association constants and the binding enthalpy pointed toward binding of the diamidines at AT regions of the DNA. The results also indicate that the diamidines bind at the outside of the DNA double helix, probably to the minor groove regions, with hydrogen bonds connecting the amide nitrogen of the diamidine to carbonyl oxygen atoms of thymidine or adenosine bases. However, CGP040215A and CGP033829A, the bisaryl diamidines, showed higher affinity than CGP039937A, the monoaryl diamidine. The spectrophotometric analysis of the interaction of these diamidines to test their effects on the melting temperature of leishmanial DNA suggests non-intercalating binding. The diamidines also showed potent inhibition of DNA polymerase activity of L. infantum extracts in vitro.     

The binding of echinomycin to deoxyribonucleic acid

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

Differential DNA recognition by the enantiomers of 1-Rh(MGP)2 phi: a combination of shape selection and direct readout

The enantiomers of the symmetric metallointercalator complex 1-Rh(MGP)2phi5+ [MGP = 4-(guanidylmethyl)-1,10-phenanthroline; phi = phenanthrenequinone diimine] bound to DNA decamer duplexes containing their respective 6 bp recognition sequences have been investigated using 1H NMR. Shape selection due to the chirality of the metal center and hydrogen-bonding contacts of ancillary guanidinium groups to 3'-G N7 atoms define the recognition by complexes which bind by intercalation to duplex DNA. The titration of Lambda-Rh into the self-complementary decamer containing the recognition sequence (5'-GACATATGTC-3', L1) resulted in one symmetric bound conformation observed in the 1H NMR spectrum, indicating that the DNA duplex retains its symmetry in the presence of the metal complex. Upfield chemical shifts of duplex imino protons and the disruption of the NOE base-sugar contacts defined the central T5-A6 intercalation site. The downfield shift of the G8 imino proton supports the conclusion that the pendant guanidinium arms make simultaneous H-bonding contacts to the N7 atoms of 3'-G8 bases on either side of the site. A variable-temperature study of a partially titrated sample (2:3 Lambda-Rh/L1) showed the exchange rate (kobs) at 298 K to be 68 s-1 and the activation barrier to exchange (DeltaG of association) to be 2.7 kcal/mol, a value comparable to the stacking energy of one base step. The results presented coupled with biochemical data are therefore consistent with binding models in which Lambda-1-Rh(MGP)2phi5+ (Lambda-Rh) traps the recognition site 5'-CATATG-3' in an unwound state, permitting intercalation centrally and hydrogen bonding to guanines at the first and sixth base pair positions. The data suggest a different model of binding and recognition by Delta-Rh. The titration of Delta-Rh into a DNA decamer containing the 6 bp recognition site (D1, 5'-CGCATCTGAC-3'; D2, 5'-GTCAGATGCG-3') resulted in two, distinct conformers, in slow exchange on the NMR time scale. The rate of exchange between the two conformers (kobs) at 298 K is 37 s-1, most likely due to partial dissociation between binding modes. The slower rate relative to Lambda-Rh association reflects the relative rigidity of the D1 and/or D2 sequence in comparison to L1. NOE cross-peaks between the intercalating phi ligand and protons of T5-C6, as well as the upfield shifts observed for imino protons at this step, serve to define the central T5-C6 step as the single site of intercalation. The downfield shift of the 3'-G imino protons indicates the complex makes hydrogen bond contacts with these bases. The complex, which is too small to span a 6 bp B-form DNA sequence, nonetheless makes major groove contacts with 3'-G bases to either side of the site. Notably, both 3'-guanine bases are necessary to impart site specificity and slow dissociation kinetics with the 5'-CATCTG-3' site, as evidenced by the extremely exchange-broadened two-dimensional NOESY spectra of Delta-Rh bound to modified duplexes containing N7-deazaguanine at either G8 or G18; the loss of one major groove contact completely abolishes specificity for 5'-CATCTG-3'. DNA chemical shifts upon binding and intermolecular NOE contacts therefore support a model in which Delta-Rh intercalates in one of two canted binding conformations. Within this model, each intercalation mode allows one guanidinium-guanine hydrogen bond at a time, while bringing the other arm close to the phosphate backbone.     

Trajectory and current situation of biomedical research in Argentina]

A cationic manganese porphyrin-peptide nucleic acid (PNA) conjugate has been prepared and used to cleave a double-stranded DNA target. Cleavage experiments were performed with a 247-base pair restriction DNA fragment containing a 10-base pair homopurine binding target for the PNA. Oxidative activation by this Mn porphyrin-PNA conjugate leads to sequence specific, 3'-staggered cleavage of both DNA strands near the strand displacement junction. Furthermore, the Mn porphyrin-PNA porphyrin conjugates bind over 100-fold better to double-stranded DNA compared to the native PNA.     

Can energy transfer be an indicator for DNA intercalation?

Contact energy transfer from DNA bases to various ligands, which can be represented by the ratio of the fluorescence intensity Q(lambda)/Q310nm, is measured by conventional fluorometer. 4',6-Diamidino-2-phenylindole and 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole can accept energy from DNA bases and exhibit the ratio Q(lambda)/Q310nm, similar to that of intercalators, although these molecules are known to bind preferentially to the minor groove of the adenine-thymine rich region of DNA. When porphyrin is intercalated in DNA or poly[d(G-C)2], the shape of the ratio Q(lambda)/Q310nm is distinct from that of the ethidium-DNA complex with two maxima at 250 nm and 280 nm. The ratio Q(lambda)/Q310nm of the porphyrin-poly[d(A-T)2] complex, in which porphyrin is known to bind 'outside' of the DNA stem, is similar to that of intercalators. Therefore, energy from excited DNA bases can be transferred not only to an intercalated ligand through direct contact but also to that bound in the minor groove. It follows from this observation that using energy transfer as a criterion for DNA intercalation requires extreme caution.     

Synthesis and binding properties of conjugates between oligodeoxynucleotides and daunorubicin derivatives

Conjugation of an anthracycline to a triplex-forming oligonucleotide (TFO) allows delivery of this drug to a specific DNA site, preserving the intercalation geometry of this class of anticancer agents. Conjugate 11, in which the TFO is linked via a hexamethylene bridge to the O-4 on the D ring of the anthraquinone moiety, affords the most stable triple helix, through intercalation of the planar chromophore between DNA bases and binding of both the TFO and the amino sugar to the major and the minor groove respectively.     

Solution conformation of the (-)-trans-anti-[BP]dG adduct opposite a deletion site in a DNA duplex: intercalation of the covalently attached benzo[a]pyrene into the helix with base displacement of the modified deoxyguanosine into the minor groove

A combined NMR-computational approach was employed to determine the solution structure of the (-)-trans-anti-[BP]dG adduct positioned opposite a -1 deletion site in the d(C1-C2-A3-T4-C5- [BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14-A15-G1 6-G17-A18-T19-G20-G21) sequence context. The (-)-trans-anti-[BP]dG moiety is derived from the binding of the (-)-anti-benzo[a]pyrene diol epoxide [(-)-anti-BPDE] to N2 of dG6 and has a 10R absolute configuration at the [BP]dG linkage site. The exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space followed by restrained molecular dynamics calculations based on a NOE distance and intensity refinement protocol. Our structural studies establish that the aromatic BP ring system intercalates into the helix opposite the deletion site, while the modified deoxyguanosine residue is displaced into the minor groove with its face parallel to the helix axis. The intercalation site is wedge-shaped and the BP aromatic ring system stacks over intact flanking Watson-Crick dG.dC base pairs. The modified deoxyguanosine stacks over the minor groove face of the sugar ring of the 5'-flanking dC5 residue. The BP moiety is positioned with the benzylic ring oriented toward the minor groove and the distal pyrenyl aromatic ring directed toward the major groove. This conformation strikingly contrasts with the corresponding structure in the full duplex with the same 10R (-)-trans-anti-[BP]dG lesion positioned opposite a complementary dC residue [de los Santos et al. (1992) Biochemistry 31, 5245-5252); in this case the aromatic BP ring system is located in the minor groove, and there is no disruption of the [BP]dG.dC Watson-Crick base pairing alignment. The intercalation-base displacement features of the 10R (-)-trans-anti-[BP]dG adduct opposite a deletion site have features in common to those of the 10S (+)-trans-anti-[BP]dG adduct opposite a deletion site previously reported by Cosman et al. [(1994)(Biochemistry 33, 11507-11517], except that there is a nearly 180 degrees rotation of the BP residue about the axis of the helix at the base-displaced intercalation site and the modified deoxyguanosine is positioned in the opposite groove. In the 10S adduct, the benzylic ring is in the major groove and the aromatic ring systems point toward the minor groove. This work extends the theme of opposite orientations of adducts derived from chiral pairs of (+)- and (-)-anti-BPDE enantiomers; both 10S and 10R adducts can be positioned with opposite orientations either in the minor groove or at base displaced intercalation sites, depending on the presence or absence of the partner dC base in the complementary strand.     

A carbamate analogue of amsacrine with activity against non-cycling cells stimulates topoisomerase II cleavage at DNA sites distinct from those of amsacrine

AMCA (methyl N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), an amsacrine analogue containing a methylcarbamate rather than a methylsulphonamide side chain, contrasts with amsacrine, doxorubicin and etoposide in its relatively high cytotoxicity against non-cycling tumour cells. AMCA bound DNA more tightly than amsacrine, but the DNA base selectivity of binding, as measured by ethidium displacement from poly[dA-dT].[dA-dT] and poly[dG-dC].[dG-dC], was unchanged. AMCA-induced topoisomerase cleavage sites on pBR322, C-MYC and SV40 DNA were investigated using agarose or sequencing gels. DNA fragments were end-labelled, incubated with purified topoisomerase II from different mammalian sources and analysed after treatment with sodium dodecylsulphate/proteinase K. AMCA stimulated the cleavage activity of topoisomerase II, but the DNA sequence selectivity of cleavage was different from that of amsacrine and other topoisomerase inhibitors. It was similar to that of the methoxy derivative of AMCA, indicating that the changed specificity resulted from the carbamate group rather than from the methoxy group. The pattern of DNA cleavage induced by AMCA was similar for topoisomerase II alpha and II beta.     

The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (DeltaOBP). We have now performed a quantitative analysis of the interaction between DeltaOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of DeltaOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with DeltaOBP in a sequence-specific manner.     

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.     

Solution structure of the aminofluorene-intercalated conformer of the syn [AF]-C8-dG adduct opposite a--2 deletion site in the NarI hot spot sequence context

This paper addresses structural issues related to the capacity of aminofluorene [AF] for frameshift mutations of the -2 type on C8 covalent adduct formation at the G3 site in the d(C-G1-G2-C-G3-C-C) NarI hot spot sequence. This problem has been approached from a combined NMR and relaxation matrix analysis computational structural study of the [AF]dG adduct in the d(C-G-G-C-[AF]G-C-C).d(G-G-C-C-G) sequence context at the 12/10-mer adduct level (designated [AF]dG.del(-2) 12/10-mer). The proton spectra of this system are of exceptional quality and are consistent with the formation of an AF-intercalated conformer with the modified guanine in a syn alignment displaced along with the 5'-flanking cytosine residue into the major groove. The solution structure has been determined by initially incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bound deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space and subsequently refined through restrainted molecular dynamics calculations based on a NOE distance and intensity refinement protocol. Strikingly, the [AF]dG.del(-2) 12/10-mer duplex adopts only one of two potential AF-intercalation alignments for the [AF]dG adduct opposite the -2 deletion site in the NarI sequence context with the extrusion of the dC-[AF]dG step favored completely over extrusion of the [AF]dG-dC step at the lesion site. This polarity establishes that the structural perturbation extends 5' rather than 3' to the [AF]dG lesion site in the adduct duplex. This structure of the [AF]dG adduct opposite a -2 deletion site shows distinct differences with conclusions reported on the alignment of the related acetylaminofluorene [AAF]dG adduct opposite a -2 deletion site in the identical NarI sequence context [Milhe, C., Fuchs, R. P. P., and Lefevre, J. F. (1996) Eur. J. Biochem. 235, 120-127]. In that study, qualitative NMR data without computational analysis were employed to conclude that the extrusion at the lesion site occurs at the [AAF]dG-dC step for the AAF-intercalated conformer of the adduct duplex. The structure of the [AF]dG adduct opposite a -2 deletion site determined in our group provides molecular insights into the architecture of extended slipped mutagenic intermediates involving aromatic amine intercalation and base-displaced syn modified guanines in AF and, by analogy, AAF-induced mutagenesis in the NarI hot spot sequence context.     

Z-->B transition in poly[d(G-m5C)2] induced by interaction with 4',6-diamidino-2-phenylindole

The Z form of poly[d(G-m5C)2], in presence of Mg2+ ion, is found to be transformed into B form upon interaction with 4',6-diamidino-2-phenylindole (DAPI). The Z-->B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6-7 base pairs. An interaction between DAPI and poly[d(G-m5C)2] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time-resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature.     

Solution conformation of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dC in a DNA duplex

Combined NMR-molecular mechanics computational studies were undertaken on the C8-deoxyguanosine adduct formed by the carcinogen 1-nitropyrene embedded in the d(C5-[AP]G6-C7).d(G16-C17-G18) sequence context in a 11-mer duplex, with dC opposite the modified deoxyguanosine. The exchangeable and nonexchangeable protons of the aminopyrene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of several proton resonances for the three nucleotide d(G16-C17-G18) segment positioned opposite the [AP]dG6 lesion site resulting in weaker NOEs involving these protons in the adduct duplex. The solution conformation of the [AP]dG.dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine ring of [AP]dG6 is displaced into the major groove and stacks with the major groove edge of dC5 in the adduct duplex. Both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove as a consequence of the aminopyrene ring intercalation into the helix. This base-displaced intercalative structure of the [AP]dG.dC 11-mer duplex exhibits several unusually shifted proton resonances which can be accounted for by the ring current contributions of the deoxyguanosinyl and pyrenyl rings of the [AP]dG6 adduct. In summary, intercalation of the aminopyrene moiety is accompanied by displacement of both [AP]dG6 and the partner dC17 into the major groove in the [AP]dG.dC 11-mer duplex.