Degradation of structurally modified DNAs by bleomycin group antibiotics

Bleomycin-mediated DNA strand scission has been shown to be diminished at certain sequences in proximity to 5-methylcytidines. We have investigated the molecular basis of this observed diminution using selective bleomycin (BLM) modifications at the C-terminus. Of the four different bleomycin congeners investigated, only bleomycin A2 and bleomycin BAPP were substantially affected by cytidine methylation. We have also examined the effect of other DNA modifications on bleomycin-mediated strand scission. Methylation at the N6 position of adenosine resulted in diminution of DNA cleavage by all four bleomycin congeners. The presence of bulky 5-(glucosyloxy)methyl groups in the major groove of T4 DNA had little effect on the efficiency of DNA strand scission mediated by bleomycin A2 or B2, suggesting the absence of important steric interactions between Fe(II).BLM and DNA in the major groove. In contrast, DNA cleavage mediated by bleomycin congeners was very sensitive to a major DNA conformational change, the B----Z transition. Salt and MgCl2 titrations of the DNA copolymers poly(dG-dC).poly(dG-dC) and poly(dG-MedC).poly(dG-MedC) demonstrated that bleomycin A2 and B2 did not cleave Z-DNA efficiently. In addition, circular dichroism titrations of these copolymers revealed that both bleomycin congeners increased the cation concentration necessary to induce the B----Z transition, implying that bleomycin preferentially binds to and stabilizes B-form DNA. These results are consistent with a model in which cytidine methylation at appropriate sequences of DNA is sufficient to induce subtle conformational changes that render the helix unreceptive to cleavage by some bleomycin congeners.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-stranded Polydeoxyribonucleotides. 3. Binding at the Minor Groove

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dGdC) · poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism signal of TPT bound to DNA was shown to have positive sign in the range 260–295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT forms an angle less than 54° with the long axis of DNA, and hence the TPT molecule cannot intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose cytosines in the major groove were all glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC) · poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.     

A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the non-covalent CC-1065/B-DNA binding

The comparative DNA binding properties and cytotoxic activity of CDPIn methyl esters (n = 1-5) vs. PDE-In methyl esters (n = 1-3) are detailed in studies which provide experimental evidence for the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the CC-1065/B-DNA minor groove binding. High affinity minor groove binding to DNA was established through: (1) the observation of CDPI3 binding (UV) but not unwinding of supercoiled DNA (phi 174 RFI DNA) thus excluding intercalative binding; (2) the observation of CDPI3 binding to T4 phage DNA (UV, delta Tm) in which the major groove is occluded by glycosylation thus excluding major groove binding; (3) the observation of salt (Na+) concentration independent high affinity CDPI3 binding to poly(dA . poly(dT) thus excluding simple electrostatic binding to the DNA phosphate backbone; and further inferred through (4) the observation of an intense induced dichroism (ICD, poly(dA) . poly(dT) and poly(dG) . poly(dC) [phi]23(358) = 24,000 and 23,500). This high affinity minor groove binding is sufficient to produce a potent cytotoxic effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxins B1 and B2 with DNA and the oligodeoxynucleotide d(ATGCAT)2

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B1 and B2 with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B2 with the oligodeoxynucleotide d(ATGCAT)2. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B1 binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin less than 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels less than 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a "two-site" binding model [L.S. Rosenberg, M.J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002-1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B2 binding to d(ATGCAT)2 indicates that the aflatoxin B2/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1-0.5 ppm are observed for the aflatoxin B2 4-OCH3, H5, and H6a protons. Much smaller chemical shift changes (less than or equal to 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T1 experiments demonstrate a 15-25% decrease in the relaxation time for the adenine H2 protons when aflatoxin B2 is added to the solution. This result suggests that aflatoxin B2 protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B1 and B2 complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B1-guanine N7 DNA adducts.     

Mode of reversible binding of neocarzinostatin chromophore to DNA: evidence for binding via the minor groove

Two general approaches have been taken to understand the mechanism of the reversible binding of the nonprotein chromophore of neocarzinostatin to DNA: (1) measurement of the relative affinity of the chromophore for various DNAs that have one or both grooves blocked by bulky groups and (2) studies on the influence of adenine-thymine residue-specific, minor groove binding agents such as the antibiotics netropsin and distamycin on the chromophore-DNA interaction. Experiments using synthetic DNAs containing halogen group (Br, I) substituents in the major groove or natural DNAs with glucosyl moieties projecting into the major groove show that obstruction of the major groove does not decrease the binding stoichiometry or the binding constant for the DNA-chromophore interaction. Chemical methylation of bases in both grooves of calf thymus DNA, resulting in 13% methylation of N-7 of guanine in the major groove and 7% methylation of N-3 of adenine in the minor groove, decreases the binding affinity and increases the size of the binding site for neocarzinostatin chromophore. Similar results were obtained whether binding parameters were determined directly by spectroscopic measurements or indirectly by measuring the ability of the DNA to protect the chromophore against degradation. On the other hand, netropsin and distamycin compete with neocarzinostatin chromophore for binding to the minor groove of DNA, as shown by their decrease in the ability of poly(dA-dT) to protect the chromophore against degradation and their reduction in chromophore-induced DNA damage as measured by thymine release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics simulations of excimer forming (+)-anti-BPDE-DNA adducts in aqueous solution

The chemical carcinogen (+)-anti BPDE preferentially binds covalently to the guanine base in the minor groove of DNA. Fluorescence spectroscopic studies have shown that the BPDE molecules bound to DNA can interact in their photo-excited state giving strong excimer fluorescence when bound to poly(dGdC) · poly(dGdC). It was suggested that the formation of such excited state complexes is most probable when the two (+)-anti-BPDE bind to guanines of adjacent base pairs on the two different strands of the DNA. In the present work a model for such an excimer forming DNA-BPDE double adduct system has been constructed and shown to be stable over a 300 ps molecular dynamics simulation in a water box. The model is a d(CG)₃ · d(CG)₃ molecule with two BPDE molecules bound to the guanines at the 4th position on each strand, located in the minor groove and each oriented towards the 5 end of the modified strand, respectively. The results of 300 ps MD simulation show that the two BPDE chromophores exhibited on the average a relative geometry favourable for excimer formation. The local structure at the adduct position was considerably distorted and the helix axis was bent. The modified bases were found to be paired through a stable single non-Watson Crick type of hydrogen bond. Correspondence to: A. Gräslund     

Molecular dynamics of spermine-DNA interactions: sequence specificity and DNA bending for a simple ligand.

We used molecular dynamics to model interactions between the physiologically important polyamine spermine and two B-DNA oligomers, the homopolymer (dG)10-(dC)10 and the heteropolymer (dGdC)5-(dGdC)5. Water and counterions were included in the simulation. Starting coordinates for spermine-DNA complexes were structures obtained by molecular mechanics modeling of spermine with the two oligomers; in these models, spermine binding induced a bend in the heteropolymer but not in the homopolymer. During approximately 40 psec of molecular dynamics simulation, spermine moves away from the floor of the major groove and interacts nospecifically with d(G)10-d(C)10. In contrast, a spermine-induced bend in the helix of (dGdC)5-(dGdC)5 is maintained throughout the simulation and spermine remains closely associated with the major groove. These results provide further evidence that the binding of spermine to nucleic acids can be sequence specific and that bending of alternating purine-pyrimidine sequences may be a physiologically important result of spermine binding.     

Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B-A transition in solution

Single-walled carbon nanotubes (SWNTs) have been considered as the leading candidate for nanodevice applications ranging from gene therapy and novel drug delivery to membrane separations. The miniaturization of DNA-nanotube devices for biological applications requires fully understanding DNA-nanotube interaction mechanism. We report here, for the first time, that DNA destabilization and conformational transition induced by SWNTs are sequence-dependent. Contrasting changes for SWNTs binding to poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were observed. For GC homopolymer, DNA melting temperature was decreased 40°C by SWNTs but no change for AT-DNA. SWNTs can induce B–A transition for GC-DNA but AT-DNA resisted the transition. Our circular dichroism, competitive binding assay and triplex destabilization studies provide direct evidence that SWNTs induce DNA B–A transition in solution and they bind to the DNA major groove with GC preference.     

Preferential protection of the minor groove of non-operator DNA by lac repressor against methylation by dimethyl sulphate.

The binding of lactose repressor to non-operator DNA was studied by the modification of several DNA's, including glycosylated DNA, with dimethyl sulphate, which affects the minor and major grooves of DNA and single stranded DNA regions. The non-specific binding of the repressor to DNA protected the minor groove but apparently not the major groove of the DNA double helix against methylation and did not increase the content of single stranded DNA regions. This suggests that the repressor on binding to non-operator DNA makes contacts mainly in the minor groove of DNA and does not uncoil the DNA double helix. This is different from the interaction of the repressor with lactose operator DNA which occurs, as shown by Gilbert et al. (1), along both the major and the minor groove.     

A molecular dynamics simulation of a polyamine-induced conformational change of DNA. A possible mechanism for the B to Z transition.

A 75ps molecular dynamics simulation has been performed on a fully solvated complex of spermine with the B DNA decamer (dGdC)5.(dGdC)5. The simulation indicates a possible mechanism by which polyamines might induce the formation of a left-handed helix, the B to Z transition. Spermine was initially located in the major groove, hydrogen bonded to the helix. During the simulation the ligand migrates deeper into the DNA, maintaining strong hydrogen bonding to the central guanine bases and destroying the Watson-Crick base pairing with their respective cytosines. Significant rotation of these and other cytosine bases was observed, in part due to interactions of the helix with the aminopropyl chains of spermine. An intermediate BII conformation might be of importance in this process.     

Structure of a beta-alanine-linked polyamide bound to a full helical turn of purine tract DNA in the 1:1 motif

Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methylhydroxypyrrole (Hp) amino acids linked by beta-alanine (beta) bind the minor groove of DNA in 1:1 and 2:1 ligand to DNA stoichiometries. Although the energetics and structure of the 2:1 complex has been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. We present here the 1:1 solution structure of ImPy-beta-Im-beta-ImPy-beta-Dp bound in a single orientation to its match site within the DNA duplex 5'-CCAAAGAGAAGCG-3'.5'-CGCTTCTCTTTGG-3' (match site in bold), as determined by 2D (1)H NMR methods. The representative ensemble of 12 conformers has no distance constraint violations greater than 0.13 A and a pairwise RMSD over the binding site of 0.80 A. Intermolecular NOEs place the polyamide deep inside the minor groove, and oriented N-C with the 3'-5' direction of the purine-rich strand. Analysis of the high-resolution structure reveals the ligand bound 1:1 completely within the minor groove for a full turn of the DNA helix. The DNA is B-form (average rise=3.3 A, twist=38 degrees ) with a narrow minor groove closing down to 3.0-4.5 A in the binding site. The ligand and DNA are aligned in register, with each polyamide NH group forming bifurcated hydrogen bonds of similar length to purine N3 and pyrimidine O2 atoms on the floor of the minor groove. Each imidazole group is hydrogen bonded via its N3 atom to its proximal guanine's exocyclic amino group. The important roles of beta-alanine and imidazole for 1:1 binding are discussed.     

Synthesis of nonlinear DNA-binding peptide with binding specificity determinants close to those of 434 Cro-repressor

Design, synthesis and DNA binding activity of a nonlinear 102 residue peptide are reported. The peptide contains four sequence-specific DNA binding domains of 434 Cro protein. These four domains were linked covalently to a symmetrical carboxyterminal crosslinker that contains four arms each ending with an aliphatic aminogroup. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha helical, beta-sheet and random coiled conformations with an alpha helical content of about 16% at room temperature. The alpha helicity is increased up to 40% in the presence of 40% trifluoroethanol. Upon complex formation between the peptide and DNA a change in the peptide conformation takes place which is consistent with an alpha-beta transition in the DNA binding, helix-turn-helix motif of 434 Cro repressor. Evidently residues present in helices alpha(2) and alpha(3) form a beta hairpin which is inserted in the minor DNA groove. The latter inference is supported by our observations that the peptide can displace minor groove binding antibiotic distamycin A from a complex with poly(dA).poly(dT). As revealed from DNase protection studies the peptide exhibits preferences for binding to operator and pseudooperator sites recognized by 434 Cro repressor. It binds strongly to operator sites OR1, OR2 and OR3 and exhibits a greater affinity for pseudooperator site Op1. From analysis of nucleotide sequences in the strong affinity binding sites for the peptide on DNA a conclusion is drawn that it binds to pseudosymmetrical nucleotide sequences 5'-ACAA(W)nCTGT-3', where W is an arbitrary nucleotide. n is equal to six or seven. In the strongest affinity binding site for the peptide on DNA (Op1) motif 5'-ACAA-3' is replaced by sequence 5'-ACCA-3'. A difference in binding specificity shown by the peptide and 434 Cro protein could be attributed to a flexibility of the connecting chains between DNA-binding domains in the peptide molecule as well as to a replacement of Thr - Ala in the alpha 2 helix. Removal of two residues from the N-terminal end of helix alpha 2 in each of the four DNA binding domains of 434 Cro present in the peptide leads to a loss of binding specificity, although the modified peptide binds to DNA unspecifically.     

Identification of binding mechanisms in single molecule-DNA complexes

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.     

The phenanthridine biguanides efficiently differentiate between dGdC, dAdT and rArU sequences by two independent, sensitive spectroscopic methods

At submicromolar concentrations two novel phenanthridine biguanides exhibit distinctly different spectroscopic signals for dGdC and dAdT sequences, respectively, by opposite fluorimetric changes (quenching for dGdC and increase for dAdT) and especially the bis-biguanide derivative gives an opposite ICD response (negative ICD for dGC and strong positive for dAdT). This specific signalling was explained by the ability of compounds to switch the binding mode from intercalation into dGdC to minor groove binding into dAdT sequences. Both compounds bind to rArU by intercalation, yielding different fluorimetric and CD response in comparison to any of aforementioned ds-DNA. Moreover, both compounds revealed significantly higher affinity toward ds-polynucleotides in comparison to previously studied alkylamine- and urea-analogues. Furthermore, DNA/RNA binding properties of novel compounds could be controlled by pH, due to the protonation of heterocyclic nitrogen. Low in vitro cytotoxicity of both compounds against human cell lines makes them interesting spectrophotometric probes.     

A role for the metal binding domain in determining the DNA sequence selectivity of Fe-bleomycin.

Previous studies of Fe-bleomycin-mediated DNA cleavage have established that the bithiazole moiety + C-terminal substituent of bleomycin are required for DNA binding, while the metal binding domain is responsible for O2 activation. Although recent studies have indicated that the metal binding domain also participates in DNA unwinding, and in determining the sequence and strand selectivity of DNA cleavage, no study has defined the structural domain that bears primary responsibility for the observed pattern of bleomycin-mediated DNA degradation. Presently, by the use of four synthetic analogs of bleomycin demethyl A2 having the functional domains connected by rigid spacers of varying lengths, the source of DNA cleavage specificity has been determined. When the four analogs cleaved 242- and 127-base pair 5'-32P-end-labeled DNA restriction fragments containing isolated Fe-bleomycin cleavage sites, all four produced cleavage at the same preferred sites. Because the (oligo)glycine spacers altered the distance between the domains by as much as 14 A, the identical cleavage patterns argue that the primary determinant of sequence specificity for these analogs is the metal binding domain.     

Determination of netropsin-DNA binding constants from footprinting data

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.     

A Molecular Dynamics Simulation of a Polyamine-Induced Conformational Change of DNA. A Possible Mechanism for the B to Z Transition

Abstract

A 75ps molecular dynamics simulation has been performed on a fully solvated complex of spermine with the B DNA decamer (dGdC)₅ · (dGdC)₅. The simulation indicates a possible mechanism by which polyamines might induce the formation of a left-handed helix, the B to Z transition. Spermine was initially located in the major groove, hydrogen bonded to the helix. During the simulation the ligand migrates deeper into the DNA, maintaining strong hydrogen bonding to the central guanine bases and destroying the Watson-Crick base pairing with their respective cytosines. Significant rotation of these and other cytosine bases was observed, in part due to interactions of the helix with the aminopropyl chains of spermine. An intermediate B_II conformation might be of importance in this process.     

Carcinogen-Nucleic Acid Interactions: Equilibrium Binding Studies of Aflatoxins B1 and B2 with DNA and the Oligodeoxynucleotide d(ATGCAT)2

Abstract

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B₁ and B₂ with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B₂ with the oligodeoxynucleotide d(ATGCAT)₂. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B₁ binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin ? 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels ? 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a “two-site” binding model [L.S. Rosenberg, M J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002–1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B₂ binding to d(ATGCAT)₂ indicates that the aflatoxin B₂/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1–0.5 ppm are observed for the aflatoxin B₂ 4-OCH₃, H5, and H6a protons. Much smaller chemical shift changes ? 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T₁ experiments demonstrate a 15–25 % decrease in the relaxation time for the adenine H2 protons when aflatoxin B₂ is added to the solution. This result suggests that aflatoxin B₂ protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B₁ and B₂ complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B₁,-guanine N7 DNA adducts.     

Quantitative analysis of DNA-porphyrin interactions

The binding of manganese(III)-tetra(4-N-methylpyridyl)porphyrin (MnTMpyP) with synthetic poly(dA-dT)2, poly(dI-dC)2, and poly(dG-dC)2 DNAs as well as calf thymus (CT) DNA has been quantitatively studied in detail using induced CD (circular dichroism) spectroscopy in the Soret absorption band. The CD spectra, which changed greatly depending on the porphyrin to DNA base-pair molar ratio (r), were normalized with respect to DNA concentration and deconvoluted. Three independent component binding modes (named mode 1, 2, and 3 in the order of increasing r values) were identified, which successfully simulated the observed CD spectra with negligibly small residuals for a wide range of r values. In the case of poly(dA-dT)2, poly (dI-dC)2, and CT DNA, all the three modes appeared, whereas in the case of poly(dG-dC)2 DNA, only modes 1 and 3 appeared in the r range studied. The r dependence of each binding mode, i.e., its relative affinity toward DNA, has been revealed by this analysis. Mode 1, which appeared as a single binding mode at very low r values (r < or = ca. 0.05), was inhibited by the addition of methyl green, a drug that preferentially binds to the major groove of poly (dA-dT)2 DNA. Berenil, a known minor groove binder to poly(dA-dT)2 or poly(dI-dC)2 DNA, inhibited modes 2 and 3. From these inhibition experiments as well as comparison of the component spectra for DNAs of different sequence, a binding site on DNA was proposed for each component binding mode. The number of DNA base pairs covered by a single molecule of porphyrin was estimated.