Interaction of Two Peptide-Acridine Conjugates Containing the SPKK Peptide Motif with DNA and Chromatin

Abstract

The interaction between DNA and two peptide-acridine conjugates containing one (1) or two (2) moieties of the Ser-Pro-Lys-Lys (SPKK) minor groove-binding peptide motif has been studied by a combination of hydrodynamic, biochemical and spectroscopic methods including diffusion-enhanced luminescence energy transfer (DELET) measurements with a Tb(III) lanthanide chelate as donor. Viscometric titrations do not reveal any significant difference between the two hybrid molecules which both unwind (by about 15°) and extend the DNA similarly. DELET measurements show that the acridinyl chromophore of compounds 1 and 2 is much more accessible than that of a simple monointercalating drug such as acridine orange or ethidium. The accessibility factor increases proportionally with the peptide length, reflecting the extent of perturbation imposed upon the intercalating chromophore by the binding to DNA of the peptide moiety of the hybrids. Experiments with the osmium tetroxide- bispyridine reagent indicate that the two hybrid compounds both affect the local conformation of DNA rendering certain thymine residues conspicuously accessible to the probe. The drug-induced sites of hyperreactivity towards OsO₄ in DNA are very similar with the exception of a short run of three T residues which is attacked more strongly in the presence of tetrapeptide-acridine conjugate 1 than with the octapeptide-acridine conjugate 2. These results are fully in agreement with previous footprinting studies and support the view that a minimum of two SPKK motifs is required to mimic the AT-specific minor groove binding antibiotic netropsin. On the basis of the DNA-binding properties of these two peptide-acridine hybrids, we present DNA-binding models in which the acridinyl moiety of compound 1 protrudes slightly outside the double helix but remains more or less parallel to the plane of the base-pairs. In contrast, with compound 2, where the octapeptide SPKKSPKK is bound to the minor groove, we postulate that the chromophore lies only partially overlapped with the base pairs in the intercalation site and, in addition, the heterocyclic chromophore is significantly tilted with respect to the double helix axis. Electric linear dichroism and DELET measurements with chromatin reveal that the presence of histone proteins affects the intercalative binding of compound 2 while it has practically no effect on the binding of compound 1.     

Energetics and stereochemistry of DNA complexation with the antitumor AT specific intercalators tilorone and m-AMSA.

Computations by the SIBFA method on the intercalative interaction energies of tilorone and m-AMSA with B-DNA representative oligonucleotides account for the specificity of these antitumor drugs for AT sites and minor groove intercalation. In tilorone this specificity is due to the strong preference of the side chains for the minor groove, which overcomes the preference of the chromophore for a GC intercalation site. In m-AMSA the specificity is due to the combined preference of both the chromophore and the anilino side chain for AT intercalation site and minor groove, respectively. o-AMSA is shown to manifest a similar (although significantly less pronounced specificity) as m-AMSA but a higher affinity for DNA. A comparison of the energetics and stereochemistry of intercalative binding to DNA of m-AMSA (AT minor groove specific) and 9-aminoacridine-4-carboxamide (GC major groove specific), which possess the same chromophore and differ only by the nature and position of the side chains, shows the possibility of important variations in the intercalative behaviour of chromophoric drugs as a function of the substituent groups attached to them.     

Specific binding of 2-amino-1,8-naphthyridine into a single guanine bulge as evidenced by photooxidation of GG doublet

Photoirradiation of 2-amino-1,8-naphthyridines in the presence of duplex DNA containing the GG doublet opposite a single bulge was examined. After hot piperidine treatment, DNA cleavage was observed preferentially at the GG opposite a single bulge. The cleavage efficiency was highly dependent on the nature of bulged base. The G cleavage at the GG opposite a single G bulge was exceptionally weak, suggesting an intercalative binding of 2-amino-1,8-naphthyridine chromophore into the GG step.     

Theoretical Design, Chemical Synthesis and Footprinting Analysis of a Novel Peptide Derivative of the Intercalator 7-H Pyridocarbazole Targeted Towards the Major Groove of DNA

Abstract In order to target the major groove of DNA, we have designed novel peptide derivatives of 7-H pyridocarbazole, which is the chromophoric ring of ditercalinium, a potent antitumor bisin- tercalator. We will present here the results obtained with a compound that has a D-Asn tethered to the pyridinium nitrogen of the ring by a protonated β-alanyl-ethyl chain. We have investigated two alternative means of intercalation of the chromophore: first, into the (pur-pur) sequences, d(CpG)(2) and d(CpA)·d(TpG); second, into the (pur-pyr) sequences, d(GpC)(2)and d(GpT)·d(ApC). For the first intercalative mode, the best bound triplet sequences are d(ACG)·d(CGT) and d(ACA) d(TGT), namely with an adenine immediately upstream from the intercalation site. In these complexes, the chromophore has its concave side in the major groove, its long axis nearly colinear with the mean long axis of the two base pairs of the intercalation site, and a bidentate H-bonded configuration occurs which involves the C=0 and NH groups of the D-Asn side chain and HN(6) and N(7) (resp.) of the adenine base upstream. One alkylammonium proton is H-bonded to N(7) of the guanine of the intercalation site, on the strand opposite to the one bearing the adenine. In the second intercalative mode, the chromophore's concave site now faces one DNA strand, and both alkylammonium protons are involved in H-bonds with N(7) and O(6) of the 3' guanine on the same strand. The peptide's complexes with sequences having A, G, or C upstream of this guanine were computed to be energetically competitive with those with the best (pyr-pur) triplets. This provides a rare example of energetically favourable drug intercalation in-between (pur-pyr) sequences as compared to the standard (pyr-pur) ones. The synthesis of this compound was performed, and a series of footprinting experiments undertaken on a total of approximately 300 nucleotides. These experiments were consistent with the inferences from the theoretical computations.     

The interaction of adriamycin and adriamycin analogues with nucleic acids in the B and A conformations.

The reinforced intercalative binding to DNA typical of adriamycin and daunomycin can still occur if there is epimerisation at C4' or if the O-methyl group is lost or if the 9-substituents are deleted or if the 4'-hydroxyl group is lost. In the latter two cases however, there is a reduction in affinity for the DNA, supporting the suggested role of the 9-hydroxyl and 4'-hydroxyl groups in secondary stabilization of the complex. Epimerisation at C-1' or at C-3' alters but does not abolish the intercalative mode of binding to DNA whereas epimerisation at C-7 precludes intercalation of the chromophore into the helix of DNA. In contrast to the interaction with the B-form found in DNA, the parent drugs do not intercalate into nucleic acids possessing the A-conformation and none of the above-mentioned structural changes will allow intercalation into A-form nucleic acids.     

NMR studies of chromomycin A3 interaction with DNA

The binding of chromomycin A3 to calf thymus DNA and poly(dG-dC) has been studied by 13C and 1H NMR with emphasis on the mode of binding, the role of Mg2+, and pH effects. The most prominent changes in the DNA base pair 13C NMR resonances upon complexation with chromomycin were observed for G and C bases, consistent with the G-C preference exhibited by this compound. Comparison of the 13C spectrum of DNA-bound chromomycin A3 with that of DNA-bound actinomycin D, a known intercalator, showed many similarities in the base pair resonances. This suggested the possibility that chromomycin A3 binds via an intercalative mechanism. 1H NMR studies in the imino proton, low-field region of the spectrum provided additional evidence in support of this binding mode. In the low-field spectrum of chromomycin A3 bound to calf thymus DNA, a small shoulder was observed on the upfield side of the G-C imino proton peak. Similarly, in the chromomycin A3 complex with poly(dG-dC), a well-resolved peak was found upfield from the G-C imino proton peak. These results are expected for ligands that bind by intercalation. Furthermore, in both the calf thymus and poly(dG-dC) drug complexes (in the presence of Mg2+) a broad peak was also present downfield (approximately 16 ppm from TSP) from the DNA imino protons. This was attributed to the C-9 phenolic hydroxyl proton on the chromomycin chromophore. Visible absorbance spectra at different pH values showed that the role of Mg2+ in the binding of chromomycin A3 to DNA is more than simple neutralization of the drug's anionic change.     

Truncated azinomycin analogues intercalate into DNA

The design and synthesis of a potentially more therapeutically-viable azinomycin analogue 4 based upon 3 has been completed. It involved coupling of a piperidine mustard to the acid chloride of the azinomycin chromophore. Both the designed azinomycin analogue 4 and the natural product 3 bind to DNA and cause unwinding, supporting an intercalative mode of binding.     

Theoretical Design,Chemical Synthesis and Footprinting Analysis of a Novel Peptide Derivative of the Intercalator 7-H Pyridocarbazole Targeted Towards the Major Groove of DNA

Abstract

In order to target the major groove of DNA, we have designed novel peptide derivatives of 7-H pyridocarbazole, which is the chromophoric ring of ditercalinium, a potent antitumor bisin- tercalator. We will present here the results obtained with a compound that has a D-Asn tethered to the pyridinium nitrogen of the ring by a protonated β-alanyl-ethyl chain. We have investigated two alternative means of intercalation of the chromophore: first, into the (pur-pur) sequences, d(CpG)₂ and d(CpA)·d(TpG); second, into the (pur-pyr) sequences, d(GpC)₂and d(GpT)·d(ApC). For the first intercalative mode, the best bound triplet sequences are d(ACG)·d(CGT) and d(ACA) d(TGT), namely with an adenine immediately upstream from the intercalation site. In these complexes, the chromophore has its concave side in the major groove, its long axis nearly colinear with the mean long axis of the two base pairs of the intercalation site, and a bidentate H-bonded configuration occurs which involves the C=0 and NH groups of the D-Asn side chain and HN₆ and N₇ (resp.) of the adenine base upstream. One alkylammonium proton is H-bonded to N₇ of the guanine of the intercalation site, on the strand opposite to the one bearing the adenine. In the second intercalative mode, the chromophore's concave site now faces one DNA strand, and both alkylammonium protons are involved in H-bonds with N₇ and O₆ of the 3′ guanine on the same strand. The peptide's complexes with sequences having A, G, or C upstream of this guanine were computed to be energetically competitive with those with the best (pyr-pur) triplets. This provides a rare example of energetically favourable drug intercalation in-between (pur-pyr) sequences as compared to the standard (pyr-pur) ones. The synthesis of this compound was performed, and a series of footprinting experiments undertaken on a total of approximately 300 nucleotides. These experiments were consistent with the inferences from the theoretical computations.     

DNA-nogalamycin interactions

The anthracycline antibiotic nogalamycin differs from the more common daunomycin-type anthracyclines by substitution on both ends of the intercalating chromophore, giving nogalamycin the approximate shape of a dumbbell. The chromophore of daunomycin is substituted on only one end. In nogalamycin, the positively charged amino sugar substituent of daunomycin is replaced by an uncharged nogalose sugar and a methyl ester group. The other end of nogalamycin, where daunomycin is unsubstituted, is fused to a bicyclo amino sugar with a positively charged dimethylamino group. Much larger DNA fluctuations are required for intercalative entry of nogalamycin than for entry of daunomycin. This report describes the X-ray crystal structure of the complex between nogalamycin and the self-complementary DNA hexamer d(me5CGTsAme5CG). The DNA contains cytosines methylated at the 5-positions and a phosphorothioate linkage at the TpA step. Nogalamycin intercalates at the terminal CpG steps and interacts with both strands in both grooves of the DNA. Large conformational adjustments in both nogalamycin and the DNA are necessary to form a stable, intercalative complex. The interactions of the bases with the nogalamycin substituents lead to sliding of bases relative to each other along the normal to Watson-Crick hydrogen bonds. The planarities of base pairs surrounding the intercalation site are distorted. The backbones of the two strands are distorted asymmetrically by nogalamycin with large deviations from standard B-DNA geometry. The complex between nogalamycin and DNA illustrates the conformational flexibility of DNA. The hydrogen-bonding interactions between nogalamycin and DNA do not suggest a sequence-specific binding of the drug, although additional secondary effects might lead to differences between various intercalation sites.     

Change of the higher order structure of DNA induced by the complexation with intercalating synthetic polymer, as is visualized by fluorescence microscopy.

The electrophoretic movement and Brownian motion of T4DNA, lambda DNA and their complexes with polyacrylamide (PAAm) via intercalative unit from 5-[(4-acryloylamino)phenyl]-3-amino-7-(dimethylamino)-2-methylp henazinium chloride were observed using fluorescence microscopy. It was found that T4DNA/PAAm complex migrates slower than T4DNA alone in gel electrophoresis, although they exhibit similar conformational change during the migration. Quantitative analyses of the translational diffusion of the lambda DNA and its complex in solution demonstrate that the DNA molecules extend due to intercalative binding of PAAm, suggesting the pseudo-grafting structure of the complex.     

Anthracycline-DNA interactions studied with linear dichroism and fluorescence spectroscopy

DNA-binding geometry and dynamics of a number of anthracyclines, including adriamycin and 4-demethoxydaunorubicin, interacting with DNA have been studied by means of linear dichroism and fluorescence techniques. The anthracycline chromophore is found to be approximately parallel to the plane of the DNA bases and to have a restricted mobility, as would be expected for an intercalative binding mode, but there are variations between different directions in the chromophore as well as between the drugs. From dichroic spectra of adriamycin in an anisotropic host of poly(vinyl alcohol), absorption components corresponding to transitions with mutually orthogonal polarizations have been resolved. These can be exploited to determine the orientations of the two chromophore axes in the DNA complex relative to the DNA helix axis. In a certain binding regime the long axis of the bound anthracycline chromophores (with the exception of 4-demethoxydaunorubicin) is found to be approximately 10 degrees closer to perpendicular to the helix axis than are the DNA bases. This demonstrates that the average base tilt is at least 10 degrees. By contrast, the short axis of the aglycon moiety is found to be tilted some 20-30 degrees from perpendicular. This may be because it is probing a base direction with a more pronounced, static or dynamic, inclination than the average in DNA. The drug orientation and the DNA orientation (reflecting flexibility) are observed to vary differently and nonmonotonically with binding ratio, suggesting specific binding and varying site geometries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of anticancer drug mitoxantrone with DNA analyzed by electrochemical and spectroscopic methods

Cyclic voltammetry coupled with different spectroscopic (UV/Vis, fluorescence and Raman) techniques were used to study the interaction of mitoxantrone (MTX), an antitumor drug, with calf thymus DNA in acetate buffer solutions (pH 4.5). The interaction of MTX with DNA could result a considerable decrease in the MTX peak currents and a hypochromic and bathochromic shift in the maximum adsorption bands of MTX as well as the emission quenching in the MTX fluorescence spectra. The variations in the electrochemical and spectral characteristics of MTX indicated MTX bind to DNA by an intercalative mode. This conclusion was reinforced by Raman data. The merely particular vibrations were affected in Raman, suggesting that only a portion of the chromophore of MTX was involved in the intercalation into DNA duplex. These studies are valuable for a better understanding the detailed mode of MTX-DNA interaction, which should be important in deeper insight into the therapeutic efficacy of MTX and design of new DNA targeted drug.     

Competition between anaerobic covalent linkage of neocarzinostatin chromophore to deoxyribose in DNA and oxygen-dependent strand breakage and base release

Treatment of poly(dA-dT) X poly(dA-dT) with the nonprotein chromophore of neocarzinostatin in the presence of sulfhydryls resulted in both direct and alkali-dependent base release, indicative of DNA sugar oxidation. Covalent chromophore-DNA adducts were also formed. Under anaerobic conditions, base release was strongly inhibited; however, adduct formation was not inhibited and in some cases was markedly enhanced. In the presence of dithiothreitol, anoxia increased adduct formation by a factor of 2, and a particularly stable adduct species was formed, which was recovered from nuclease digests of the treated DNA as a highly fluorescent compound with structure chromophore-d(TpApT). Acid hydrolysis of chromophore-d(TpApT) released free adenine base and both 3'dTMP and 5'dTMP, leaving a compound that contained only chromophore and the deoxyadenosine sugar. These results conclusively confirm that the chromophore forms a covalent adduct with deoxyribose in DNA. Thus, even in the absence of oxygen, activation of the chromophore by sulfhydryls results in the formation of a species capable of reacting with deoxyribose. Several other adduct species were also formed, some of which were nonfluorescent and relatively hydrophilic, but all of which were produced in increased amounts under anoxia. This inverse relation between sugar oxidation and adduct formation suggests that the two lesions share a common precursor. In the presence of other thiols, the effects of anoxia were somewhat different. With glutathione, anoxia markedly enhanced adduct formation, but the total adduct formed was considerably less than with dithiothreitol. With 2-mercaptoethanol, anoxia had no effect on total adduct formation, but the distribution of adduct species was altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a pterin derivative in Escherichia coli DNA photolyase

DNA photolyase from Escherichia coli contains reduced flavin adenine dinucleotide plus a second chromophore, partially characterized in previous studies. Both chromophores function as sensitizers in catalysis. The second chromophore has been identified as a 6-substituted pterin derivative. The compound is oxidized with permanganate to yield 6-carboxypterin or reduced with sodium cyanoborohydride to yield a 5,6,7,8-tetrahydropterin derivative. The second chromophore exhibits spectral properties (lambda max = 360, 255 nm, pH 2) similar to that observed for 7,8-dihydropterin cations. The compound does not exhibit a spectrally detectable pKa around 4 but is converted to a dication (lambda max = 346, 255 nm) in strong acid (pKa approximately 1). Similar ionization behavior is observed with 7,8-dihydropterin derivatives that are alkylated at N(5). The instability of the second chromophore in weakly alkaline solution is due to a fully reversible conversion to a labile bleached form. As compared with other pterin derivatives, the hydrolytic instability is unusual but is very similar to that observed for 5,6-dialkyl-7,8-dihydropterinium salts. It is proposed that the second chromophore is a 7,8-dihydropterin with substituents at positions 5 and 6. The discovery that a pterin derivative functions as a photosensitizer in DNA repair is apparently the first example of a photobiological function for pterins.     

Cytotoxic activities and DNA binding properties of 1-methyl-7H-indeno[1,2-b]quinolinium-7-(4-dimethylamino) benzylidene triflate

The interaction of calf thymus DNA (ct-DNA) with a novel synthesized pyrazolo[1,5-a]indole compound 1-methyl-7H-indeno[1,2-b]quinolinium-7-(4-dimethylamino) benzylidene triflate (MIDBT) was extensively studied by various spectroscopic techniques, viscosity measurements, and gel electrophoresis. The UV-visible observation implied that the compound interacted with ct-DNA by two binding modes, intercalating into the DNA base pairs and attaching to the helix exterior of DNA. The results of the fluorescent quenching and viscosity measurements showed that MIDBT could intercalate into DNA base pairs deeply in a classical intercalative mode. Circular dichroism results showed that the binding of MIDBT shifted ct-DNA conformation from B to A at low concentrations. In the gel electrophoresis, the compound was found to promote the cleavage of plasmid pBR 322 DNA effectively. Furthermore, cytotoxic studies of this compound against eleven selected tumor cell lines have been done. The values of 50% cytotoxic concentration (IC(50)) were in the range of 1.09-18.84?μM, exhibiting the potent cytotoxic properties.     

Groove selectivity in the interaction of 9-aminoacridine-4-carboxamide antitumor agents with DNA

Theoretical quantitative evaluation of the intercalative binding to DNA of the new antitumor drug 9-aminoacridine-4-carboxamide indicates that, in contradiction with a recently proposed model, the compound should show specificity for interaction with the major (and not minor) groove of GC sequences.     

Molecular structure of antitumor drug steffimycin and modelling of its binding to DNA.

The molecular and crystal structure of steffimycin have been determined by single crystal X-ray diffraction to 0.9 angstrom resolution. The triclinic crystals are in the space group P1, with the unit cell dimensions of a = 8.606(3) angstrom, b = 22.168(7) angstrom, c = 8.448(2) angstrom, alpha = 97.56(3) degrees, beta = 95.97(2) degrees, gamma = 87.94(3) degrees, Z = 2. The structure was solved by direct methods and refined by the full-matrix least-squares method to a final R value of 0.065 with 3405 (Inet greater than 2.0 sigma (Inet] observed reflections using the NRCVAX software package. The crystal lattice includes 2 independent steffimycin, 3 water and one 2-methyl-2,4-pentanediol molecules. The conformation of steffimycin is grossly similar to other anthracycline antibiotics including daunorubicin. The crystal packing interactions of steffimycin suggest a preferred stacking of the aglycone chromophore of the antibiotic which resembles the intercalative interactions seen in the daunorubicin-d(CGTACG) (Wang et al., Biochemistry 26, 1152 (1987] and nogalamycin-d(CGT(pS)ACG) (Liaw et al., Biochemistry 28, 9913 (1989] complexes. The atomic coordinates data from these complexes were used to model the intercalative binding of steffimycin to DNA. The models were then stereochemically idealized by the constraint refinement program NUCLSQ. Subsequently XPLOR software package was used for energy minimization of these models in vacuo. The model building studies suggest that steffimycin has a higher CpG base sequence specificity over the TpA step, similar to that of daunorubicin and nogalamycin.     

Binding of the nonprotein chromophore of neocarzinostatin to deoxyribonucleic acid

The methanol-extracted, nonprotein chromophore of neocarzinostatin (NCS), which has DNA-degrading activity comparable to that of the native antibiotic, was found to have a strong affinity for DNA. Binding of chromophore was shown by (1) quenching by DNA of the 440-nm fluorescence and shifting of the emission peak to 420 nm, (2) protection by DNA against spontaneous loss of activity in aqueous solution, and (3) inhibition by DNA of the spontaneous generation of 490-nm fluorescence. Good quantitative correlation was found between these three methods in measuring chromophore binding. There was nearly a 1:1 correspondence between loss of chromophore activity and generation of 490-nm fluorescence, suggesting spontaneous degradation of active chromophore to a highly fluorescent product. Chromophore showed a preference for DNA high in adenine + thymine content in both fluorescence quenching and protection studies. NCS apoprotein, which is known to bind and protect active chromophore, quenched the 440-nm fluorescence, shifted the emission peak to 420 nm, and inhibited the generation of 490-nm fluorescence. Chromophore had a higher affinity for apoprotein than for DNA. Pretreatment of chromophore with 2-mercaptoethanol increased the 440-nm fluorescence seven-fold and eliminated the tendency to generate 490-nm fluorescence. The 440-nm fluorescence of this inactive material was also quenched by DNA and shifted to 420 nm, indicating an affinity for DNA comparable to that of untreated chromophore. However, its affinity for apoprotein was much lower than that of untreated chromophore. Both 2-mercapto-ethanol-treated and untreated chromophore unwound supercoiled pMB9 DNA, suggesting intercalation by both molecules. Since no physical evidence for interaction of native neocarzinostatin with DNA has been found, it is likely that dissociation of the chromophore from the protein and association with DNA are important steps in degradation of DNA by neocarzinostatin.     

Binding of quinoline analogues of echinomycin to deoxyribonucleic acid. Role of the chromophores.

Two novel antibiotics were isolated, designated compounds 1QN and 2QN respectively, having quinoline rings in place of one or both of the quinoxaline chromophores of echinomycin. Each removes and reverses the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs. For compound 1QN, the unwinding angle at I0.01 is almost twice that of ethidium, whereas for compound 2QN the value is indistinguishable from that of ethidium. Binding of both analogues produced changes in the viscosity of sonicated rod-like DNA fragments corresponding to double the helix extension found with ethidium, a feature characteristic of bifunctional intercalation by quinoxaline antibiotics. These results suggest that both compounds 1QN and 2QN behave as bifunctional intercalators but that compound 2QN produces only half the helix unwinding seen with compound 1QN and the natural quinoxalines. Binding curves for the interaction of both analogues with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis. Values for compound 2QN were also obtained by a fluorimetric method and found to agree well with the solvent-partition measurements. Compound 1QN bound most tightly to Micrococcus lysodeikticus DNA and, like echinomycin, exhibited a broad preference for (G + C)-rich DNA species. For compound 2QN no marked (G + C) preference was indicated, and the tightest binding among the natural DNA species studied was found with DNA from Escherichia coli. The two analogues also displayed different patterns of specificity in their interaction with synthetic nucleic acids. Compound 2QN bound to poly(dA-dT) slightly more tightly than to poly-(dG-dC), whereas compound 1QN displayed a large (approx. 11-fold) preference in the opposite sense. There was evidence of co-operativity in the binding to poly(dA-dT). It may be concluded that the chromophore moieties play an active role in determining the capacity of quinomycin antibiotics to recognize and bind selectively to specific sequences in DNA.     

Free radical mechanisms in neocarzinostatin-induced DNA damage

The molecular mechanisms by which the antitumor protein antibiotic, neocarzinostatin, interacts with DNA and causes DNA sugar damage is discussed. Physical binding of the nonprotein chromophore of neocarzinostatin to DNA, involving an intercalative process and dependent on the microheterogeneity of DNA structure, is followed by thiol activation of the drug to a probable radical species. The latter attacks the deoxyribose, especially at thymidylate residues, by abstracting a hydrogen atom from C-5' to generate a carbon-centered radical on the DNA. This nascent form of DNA damage either reacts with dioxygen to form a peroxyl radical derivative, which eventuates in a strand break with a nucleoside 5'-aldehyde at the 5'-end or reacts with the bound drug to form a novel drug-deoxyribose covalent adduct. Nitroaromatic radiation sensitizers can substitute for dioxygen, but the DNA damage products are different. Similarities between the various biological effects of neocarzinostatin and ionizing radiation are reviewed.