Probing DNA single strands for single-base bulges with neocarzinostatin chromophore.

Neocarzinostatin chromophore (NCS-Chrom) induces strong cleavage at a single site (C3) in the single-stranded and 5' (32)P-end-labeled 13-mer GCCAGATTTGAGC in a reaction dependent on a thiol. By contrast, in the duplex form of the same 13-mer, strand cleavage occurs only at the T and A residues, and C3 is not cleaved. To determine the minimal structural requirement(s) for C3 cleavage in the single-stranded oligomer, several deletions and mutations were made in the 13-mer. A 10-mer (GCCAGAGAGC) derived from the 13-mer by deletion of the three T residues was also cleaved exclusively at C3 by NCS-Chrom, generating fragments having 5' phosphate ends. That the cleavage at C3 is initiated by abstraction of its 5' hydrogen is confirmed in experiments using 3' (32)P-end-labeled 10-mer. The competent 13-mer and 10-mer were assigned hairpin structures with a stem loop and a single bulged out A base, placing C3 across from and 3' to the bulge. Removal of the bulged A base from the 13-mer and the 10-mer resulted in complete loss of cutting activity, proving that it is the essential determinant in competent substrates. Studies of thiol post-activated NCS-Chrom binding to the DNA oligomers show that the drug binds to the bulge-containing 13-mer (K(d) = 0.78 microM) and the 10-mer (K(d) = 1.11 microM), much more strongly than to the 12-mer (K(d) = 20 microM) and the 9-mer (K(d) = 41 microM), lacking the single-base bulge. A mutually induced-fit between NCS-Chrom and the oligomer resulting in optimal stabilization of the drug-DNA complex is proposed to account for the site-specific cleavage at C3. These studies establish the usefulness of NCS-Chrom as a probe for single-base bulges in DNA.

DNA damage by thiol-activated neocarzinostatin chromophore at bulged sites.

Bulge structures in nucleic acids are of general biological significance and are potential targets for therapeutic drugs. It has been shown in a previous study that thiol-activated neocarzinostatin chromophore is able to cleave duplex DNA selectively at a position opposite a single unpaired cytosine or thymine base on the 3' side. In this work, we studied in greater detail the nature of this type of cleavage and the basis for the selectivity of the bulge site cleavage over the usual strand cleavage at a T site in the duplex region by using duplexes containing an internal control and a bulge, which is composed of different types and number of bases. Experimental results indicated that the bulge-induced cleavage is initiated by 5' hydrogen abstraction and is greatly affected by the base composition of the bulge. A single-base bulge, especially when containing a purine, yields higher efficiency and greater selectivity for the bulge-induced cleavage. In particular, a single adenine base gives rise to the highest cleavage yield and provides over 20 times greater selectivity for cleavage at the bulge site compared with the internal control site in duplexes. The binding dissociation constants of postactivated drug for a stem-loop structure containing a one- or two-base bulge in the stem, measured by fluorescence quenching, show that the binding is about 3-4 times stronger for bulge-containing duplexes than for perfect hairpin duplexes. For RNA.DNA hybrid duplexes, where the DNA is the target strand and the RNA is the bulge-containing strand, bulge-induced cleavage was observed, although at low yield. On the other hand, when RNA is the nonbulge strand, no bulge-induced cleavage was found. When the reaction is performed in the absence of oxygen, the major product is a covalent adduct, and it is at the same location as the cleavage site under aerobic conditions.

Mechanistic studies on the base-catalyzed transformation of neocarzinostatin chromophore: roles of bulged DNA.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore (NCS-chrom) in a base-catalyzed, radical-mediated reaction. Studies designed to elucidate the detailed mechanism of the base-catalyzed activation of NCS-chrom and to evaluate the roles of bulged DNA in its activation are described. They show that nucleobases in the DNA bulge are not required to form an effective bulge pocket but enhance the binding of the wedge-shaped activated drug molecule. Analysis of solvent deuterium isotope effects on NCS-chrom degradation and DNA cleavage efficiency experiments suggests that the spirolactone biradical 6 is a relatively stable species and that intramolecular quenching of the C2 radical of 6 to form the biologically active cyclospirolactone radical 7a occurs first (pathway a in Scheme 2), leaving the C6 radical to abstract the hydrogen atom from the DNA deoxyribose and to form the cyclospirolactone 8. Binding of the activated drug at the bulge site is required, but not sufficient, for efficient 8 formation, whereas cleavage of bulged DNA is not essential. Efficient generation of 8, but inefficient DNA damage generation, comes mainly from the likely high off-rate of 7a binding. The finding that thymidine 5'-carboxylic acid-ended oligonucleotide fragment can be formed in the reaction suggests that the process of DNA cleavage is rather slow and that sequential oxidations of the target 5'-carbon are possible. Study of the effect of solvent (methanol) concentration on NCS-chrom degradation indicates that bulged DNA acts to assist the intramolecular quenching of the radical at C2 by C8' ' of the naphthoate moiety by excluding solvent from the binding pocket, thus preventing the formation of spirolactones 9, and by blocking radical polymerization. Because in the absence or near absence of solvent methanol 8 formation does not reach even 10% that formed in the presence of bulged DNA, it is possible that the DNA bulge also induces a conformational change in the drug to promote the intramolecular reaction.

Replication block by an enediyne drug-DNA deoxyribose adduct.

Under anaerobic conditions neocarzinostatin chromophore, an enediyne antibiotic, forms a covalent drug-DNA adduct on the 5' carbon of deoxyribose at a specific single site in a 2-nucleotide bulge, rather than strand cleavage, by a mechanism involving general base-catalyzed intramolecular drug activation to a reactive radical species. We have taken advantage of the selectivity of this reaction to prepare a single-stranded oligonucleotide containing a single drug adduct at a T residue and to study its effect on the template properties of the oligonucleotide in replicative synthesis, as followed by 5'-32P-labeled primer extension by several DNA polymerases. With the Klenow fragment of Escherichia coli DNA polymerase I, synthesis stops at the base immediately 3' to the adduct. The same enzyme, but lacking 3' to 5' exonuclease activity, permits synthesis to proceed by one additional nucleotide. This effect is enhanced when Mn2+ is substituted for Mg2+. T4, herpes simplex virus, and cytomegalovirus DNA polymerases all act like Klenow polymerase. Sequenase (exo-minus T7 DNA polymerase) is qualitatively similar to exo-minus Klenow polymerase but is more efficient in inserting a nucleotide opposite the lesion. With the small-gap-filling human DNA polymerase beta, which lacks intrinsic exonucleolytic activity, primer extension proceeds to the nucleotide opposite the lesion. However, when a gap was created opposite the lesion, polymerase beta adds as many as two additional nucleotides 5' to the adduct site. The fidelity of base incorporation opposite the lesion was not impaired, in contrast with adducts on DNA bases.

Synthesis and DNA binding of spirocyclic model compounds related to the neocarzinostatin chromophore.

[formula: see text] Spirocyclic model compounds which mimic the molecular architecture of one of the decomposition products of the antitumor agent NCS-chrom have been synthesized. These readily accessible molecules bind with remarkable efficiency to bulged DNA oligonucleotides, offering potential for the design of therapeutic agents.

Characterization of a novel covalent monoadduct on the RNA overhang of an RNA-DNA hybrid induced by antitumor antibiotic neocarzinostatin.

Neocarzinostatin chromophore (NCS-chrom) has been found previously to induce thiol-dependent abasic site formation and cleavage on the RNA strand of RNA-DNA hybrids. When a four-base nucleotide was deleted from the 5' end of the DNA strand, however, an unexpected product with slow mobility, potentially a drug adduct or interstrand cross-linked material, was observed instead. End-labeling of the RNA and DNA strands showed that only the RNA strand is present in the product. The product, isolated from a denaturing polyacrylamide gel, has a fluorescence spectrum similar to that of activated NCS-chrom, and thiols with different charges give rise to products with various mobilities on the gel, indicating that the product consists of a drug adduct with NCS-chrom and thiol attached to the RNA strand of the hybrid. Mass spectroscopic (MS) analysis shows that it is a monoadduct. Chemical sequencing of 5'- and 3'-end-labeled monoadduct and MS/MS data show that drug is attached only to the U9 of the RNA in the hybrid 5'-r(CACAGAAUU9CG)/5'-d(TTCTGTG). Unlike most other DNA adducts and cross-linked products found previously, this adduct can be formed readily in the presence of atmospheric oxygen. 2'-O-Methyl and 2'-H substitutions and nucleotide replacements on the 4-base RNA overhang affect monoadduct formation to different extents, reflecting the existence of a tight three-dimensional binding pocket for the activated drug. Stability of drug adduct to heat and aniline-HOAc treatment suggest that the drug is covalently bound to the ribose of U9. Since no deuterium isotope selection effect was observed for C-1' of U9, it is possible, although not required, that the drug abstracts H from another hydrogen source on the sugar of the targeted ribonucleotide.

Characterization of a covalent monoadduct of neocarzinostatin chromophore at a DNA bulge.

Neocarzinostatin chromophore (NCS-Chrom) induces highly efficient site-specific strand cleavage at the bulge of a folded single-stranded 31-mer DNA in the presence of oxygen [Kappen, L. S., and Goldberg, I. H. (1993) Science 261, 1319-1321]. Under anaerobic conditions, the major product is a material having gel mobility slower than that of the parent 31-mer. In order to characterize this product, it was stabilized by reduction with borane/pyridine, labeled with 32P at its 5' or 3' end, and subjected to chemical cleavage dependent on base elimination or modification, and the cleavage products were analyzed on a sequencing gel. A cleavage pattern comparable to that of the 31-mer was obtained until the bases on either side of T22 at the bulge. Cleaved fragments inclusive of T22 from the 5' or the 3' end had retarded and anomalous mobilities and appeared as a smear of bands closer to the starting material, presumably due to the presence of the covalently bound drug. Pyrimidine-specific agents such as hydrazine and potassium permanganate, but not the DNA sugar-specific probe thiol-activated NCS-Chrom, induced strand cleavage at T22. Mass spectral analysis of the presumed adduct isolated from anaerobic reactions containing NCS-Chrom and a bulge duplex substrate made up of a 10-mer and an 8-mer showed that the adduct contains one molecule of the drug and one molecule of the 10-mer. Taken together, the results show that (i) drug adduction is at T22 on the full-length substrate; (ii) the pyrimidine ring is accessible to base-specific chemical modifications, hence, presumably free of the drug; (iii) it is most likely that drug adduction is via its C6 position to the 5' carbon of T22, based on the current results and the known chemistry of the hydrogen abstraction by the drug in the presence or absence of oxygen; (iv) there is no involvement of the neighboring bases by way of inter- or intrastrand cross-linking; and (v) the product is a monoadduct.

Mechanism of formation of novel covalent drug.DNA interstrand cross-links and monoadducts by enediyne antitumor antibiotics.

The potent enediyne antitumor antibiotic C1027 has been previously reported to induce novel DNA interstrand cross-links and drug monoadducts under anaerobic conditions [Xu et al. (1997) J. Am. Chem. Soc. 119, 1133-1134]. In the present study, we explored the mechanism of formation of these anaerobic DNA lesions. We found that, similar to the aerobic reaction, the diradical species of the activated drug initiates anaerobic DNA damage by abstracting hydrogen atoms from the C4', C1', and C5' positions of the A1, A2, and A3 nucleotides, respectively, in the most preferred 5'GTTA1T/5'ATA2A3C binding sequence. It is proposed that the newly generated deoxyribosyl radicals, which cannot undergo oxidation, likely add back onto the nearby unsaturated ring system of the postactivated enediyne core, inducing the formation of interstrand cross-links, connecting either A1 to A2 or A1 to A3, or drug monoadducts mainly on A2 or A3. Comparative studies with other enediynes, such as neocarzinostatin and calicheamicin gamma 1I under similar reaction conditions indicate that the anaerobic reaction process is a kinetically competitive one, depending on the proximity of the drug unsaturated ring system or dioxygen to the sugar radicals and their quenching by other hydrogen sources such as solvent or thiols. It was found that C1027 mainly generates interstrand cross-links, whereas most of the anaerobic lesions produced by neocarzinostatin are drug monoadducts. Calicheamicin gamma 1 (1) was found to be less efficient in producing both lesions. The anaerobic DNA lesions induced by enediyne antitumor antibiotics may have important implications for their potent cytotoxicity in the central regions of large tumors, where relative anaerobic conditions prevail.

Effect of ribonucleotide substitution on nucleic acid bulge recognition by neocarzinostatin.

Bulged RNA structures are not as good substrates for cleavage by the enediyne antibiotic neocarzinostatin chromophore in the general base-catalyzed reaction as are DNA bulges. In an effort to determine why this is so, we have systematically substituted ribonucleotide residues in a DNA bulged structure (CCGATGCG.CGCAGTTCGG) (cleaved residue is underlined) known to be an excellent substrate. It was found that ribonucleotide substitution at the bulge target site, as well as at other regions involving duplex formation had a small effect on the cleavage reaction, unless either of the two strands was entirely of the ribo form. By contrast, changing the A.T base pair on the 5' side of the target nucleotide (T residue) to the ribo A.U resulted in an 87% decrease in cleavage; in fact, conversion of the A alone to the ribo form caused a 68% loss in cleavage. This result can be understood from the recent solution structure of the complex formed between an analogue of the drug radical species and a bulged DNA (Stassinopoulos, A.; Ji, J.; Gao, X.; Goldberg, I.H. Science 1996, 272, 1943), since the 2' hydroxyl group of the ribo A would be expected to clash sterically with the 7"-O-methyl moiety of the drug. Additional studies on substrate bulge-dependent drug product formation and protection against spontaneous drug degradation support the cleavage experiments, and imply that bulge-specific drug binding is required for efficient cleavage.

Solution structure of a two-base DNA bulge complexed with an enediyne cleaving analog.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 +/- 0.1 angstroms from the pro-S H5' of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.

Binding specificity of post-activated neocarzinostatin chromophore drug-bulged DNA complex studied using electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was employed to characterize the binding specificity of a bulged 22-mer DNA hairpin with a post-activated neocarzinostatin chromophore (NCS-Chrom) having two similar forms, where 2a has an H in a location for which 2b has it replaced by a CH2OH group. Specific binding of 2a to the bulged 22-mer DNA was observed whereas little binding was detected for 2a to non-bulged DNA 19-mer and 12-mer duplexes. The stoichiometry of the 22-mer DNA complex with 2a was determined to be predominantly 1:1. Substitution of hydrogen in 2a for the hydroxymethylene group in 2b dramatically reduced the binding strength to the 22-mer DNA. Little complex formation was observed for 2b and 22-mer DNA based upon the ESI-MS data, consistent with earlier fluorescence studies. The results indicate that ESI-MS can be a sensitive technique for probing conformational specificity in studies of biomolecular binding.

Probing the structure of long single-stranded DNA fragments with neocarzinostatin chromophore. Extension of the base-catalyzed bulge-specific reaction.

The base-catalyzed (bc) thiol-independent cleavage reaction of neocarzinostatin chromophore (NCS chrom) has been characterized with long single-stranded (ss) DNA in order to use this reaction as a selective probe for the tertiary structure of naturally occurring ss nucleic acids. The ss circular phi chi 174 phage and M13mp18 phage DNAs (approximately 5000 and 7500 bases, respectively) were shown to be bc NCS chrom reaction substrates, exhibiting the expected pH dependence. The ss DNA fragments (150-450 bases) were cleaved at six major sites; the lesions occurred at T-rich non-double-stranded sequences, as predicted from comparison with the minimal energy secondary structures. These sites exhibited the expected pH and drug: DNA ratio dependence shown to be required for this reaction. Optimization of the shortest sequence, which gave the highest cleavage yield, identified the minimal sequence requirements for the site (19-mer of the sequence 3'TACTGAGTCTCCTTTTGTA5', attacked residue in bold). Folding pattern analysis predicted that the oligonucleotide contained a two-base bulge at the cleavage site; this result was consistent with the observation that removing features which destabilize the bulged structure increased the cleavage yield. Furthermore, the derived 19-mer was shown to generate maximal amounts of the final drug product of the bc DNA cleavage reaction. Reaction of an RNA 339-mer containing the same sequence as one of the long ss DNA fragments showed it not to be a substrate for the bc reaction, while similar results were obtained for the RNA analog of shorter oligodeoxyribonucleotides identified in this and earlier studies. Through a combination of thermodynamic and kinetic assays, the observed difference in reactivity was shown to be the result of the low binding of the cleaving species to RNA.

Double-stranded damage of DNA.RNA hybrids by neocarzinostatin chromophore: selective C-1' chemistry on the RNA strand.

Glutathione-activated neocarzinostatin chromophore generates bistranded lesions in the hybrid formed by yeast tRNA(phe) and DNA complementary to its 31-mer 3' terminus. To elucidate the chemistry of the RNA cleavage reaction and to show that the lesions are double-stranded (ds), a series of shorter oligoribonucleotides containing the target sequence r(AGAAUUC).(GAATTCT) (underlining indicates major attack site) was studied as substrates. In addition to cleavage at both U residues, major damage was produced in the form of an abasic site at the U residues. Evidence for abasic site formation on the RNA strand was obtained from sequencing-gel analysis and measurement of uracil base release. Initial evidence for the ds nature of the damage came from experiments in which 2'-O-methyluridine was substituted for uridine in the RNA at one or both of the target sites. The site containing the substitution was not a target for cleavage or abasic site formation, and the particular T residue, staggered two nucleotides in the 3' direction on the complementary DNA strand, was cleaved significantly less. These studies were valuable in identifying the DNA ds partner of the RNA attack site. Direct evidence for ds lesions came from analysis of the products from a hairpin oligonucleotide construct in which the RNA and DNA strands were linked by four T residues and contained an internal 32P label at the 3' end of the RNA strand. Substitution of deuterium for hydrogen at the C-1' position of the U residues led to a substantial isotope effect (k1H/k2H = 3) upon the formation of the RNA abasic lesion and the RNA cleavage products, providing conclusive evidence for selective 1' chemistry. On the other hand, cleavage at the T residues on the complementary DNA strand involved C-5' hydrogen abstraction, as was also true for the T residue in an oligodeoxynucleotide analogue of the RNA strand. Chemical mechanisms to account for the RNA cleavage and abasic site formation via C-1' hydrogen abstraction are proposed.

A single binding mode of activated enediyne C1027 generates two types of double-strand DNA lesions: deuterium isotope-induced shuttling between adjacent nucleotide target sites.

It has been previously reported that the potent enediyne antitumor antibiotic C1027 chromophore produces in DNA restriction fragments double-strand lesions at the sequence GTTA1T/ATA2A3C (damage positions are numbered), involving A1 and A3 [Xu, Y.-j., Zhen, Y.-s., & Goldberg, I. H. (1994) Biochemistry 33, 5947-5954]. Using oligodeoxynucleotide substrates, an additional double-strand lesion has been found within this sequence to involve A1 and A2. The lesions, which include strand breaks and abasic sites, are due to hydrogen atom abstraction from C4' at A1 and from C5'at A3 or C1' at A2 by the diradical species of activated drug. Lesions at A2 or A3 are always part of double-strand lesions. The drug radical center involved in attack at A2 or A3 is readily quenched by solvent methanol so as to produce a single-strand lesion at A1. By using methanol containing carbon-bound deuterium, there is a substantial isotope effect on the quenching reaction, resulting in enhanced double-strand lesion formation. In the absence of methanol, almost all damage at A1 belongs to double-strand lesions. There is a considerable flexibility of the drug radical attacking A2 or A3, such that the presence of deuterium at C1' of A2 results in substantial shuttling of the attack to the C5' of the neighboring nucleotide A3. These data strongly suggest the presence of a single mode of binding of activated drug but one which permits the drug diradical center attacking A2 or A3 to have considerable leeway in target selection. Quantitative affinity cleavage binding analysis is consistent with this proposal.

Binding and cleavage characteristics of the complexes formed between the neocarzinostatin chromophore and single site containing oligonucleotides.

It is shown by fluorescence spectroscopy that the post-activated form of neocarzinostatin chromophore (NCSi-glu) can form stable complexes with single-site oligonucleotides (SSOs) featuring sequences known to be involved in double stranded (AGC.GCT, AGT.ACT, AGA.TCT, ACA.TGT) or single stranded (AGG.CCT) cleavage (attacked residues in bold). Furthermore, the same SSOs form cleavage productive complexes with native neocarzinostatin chromophore (NCS chrom) over a similar concentration range. The productive complexes yield damage similar to that observed if the same sequence is part of a longer DNA piece. Previously identified double stranded site sequences ATT.AAT and TAT.ATA are shown to contain overlapping attack sites. Binding order preference derived from fluorescence quenching experiments for NCSi-glu is consistent with constants derived by quantitative cleavage affinity binding experiments with NCS chrom. This confirms the similarity in interactions between the NCSi-glu and NCS chrom and justifies the use of NCSi-glu as a stable analog of NCS chrom.

NMR studies of the post-activated neocarzinostatin chromophore-DNA complex. Conformational changes induced in drug and DNA.

The glutathione post-activated neocarzinostatin chromophore (NCSi-glu)-DNA complex was studied in detail by 2-D NMR spectroscopy. The complex is a model for understanding the sequence specific cleavage of DNA by the native neocarzinostatin chromophore (NCS chrom), a highly potent enediyne antitumor agent. NMR spectral analysis is presented for the free NCSi-glu, the free DNA duplex and the NCSi-glu-DNA complex. In addition to the previously reported structural details of the complex (Gao, X.; Stassinopoulos, A.; Rice, J. S.; Goldberg, I. H. Biochemistry 1995, 34, 40), we demonstrate that the binding of NCSi-glu in minor groove results in a patch of negatively charged surface covering the otherwise relatively neutral minor groove. The formation of the complex is largely driven by hydrophobic forces and the solvation of the polar surface of the complex. Comparison of the conformations of NCSi-glu and DNA duplex in their free and bound form reveals an induced mutual fit of DNA and NCSi-glu upon complex formation. The reduced NCS chrom represents a DNA binding motif for sequence specific recognition of DNA via intercalation and minor groove interactions.

Bulge-specific cleavage in transactivation response region RNA and its DNA analogue by neocarzinostatin chromophore.

On the basis of the finding that in the absence of thiol the nonprotein chromophore of the antitumor drug neocarzinostatin (NCS-chrom) induces highly efficient site-specific cleavage at a single site on the 3' side of a bulge in single-stranded DNA involving entirely 5' chemistry [Kappen, L. S., & Goldberg, I. H. (1993) Science, 261, 1319-1321], transactivation response region (TAR) RNA (29-mer) and its DNA analogue which presumably contain bulge structures were tested as potential substrates for NCS-chrom. In TAR RNA NCS-chrom generates a distinct but weak band due to cleavage at U24 in the bulge. Cleavage at U24 has a pH dependence and time course similar to those for previously studied DNA bulges. This band is not produced in drug reactions containing glutathione, by the protein component of native NCS, or by inactivated NCS-chrom. Cleavage at U24, albeit weak, occurs in an RNA substrate made up of two linear RNA oligomers which presumably can form a bulge akin to that in TAR RNA. In the DNA analogue of TAR RNA, as well as in a DNA duplex made of two linear oligomers that can form a similar bulge, NCS-chrom causes strand cleavage at the T residues in the bulge and at the bases flanking the bulge. Cleavage at T25 in the bulge involves, in addition to 5' chemistry, 4' attack which results in a fragment with mobility characteristic of 3'-phosphoglycolate-ended fragments. Experiments using DNA substrate having deuterium selectively at the 4' or 5' positions of T25 confirm 4' attack and show kinetic shuttling between the two positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific binding of the biradical analog of neocarzinostatin chromophore to bulged DNA: implications for thiol-independent cleavage.

The enediyne anticancer antibiotic neocarzinostatin chromophore generates a single, site-specific break at a bulge in DNA in a thiol-independent reaction, involving intramolecular drug activation under general base catalysis [Kappen, L. S., & Goldberg, I. H. (1993) Biochemistry 32, 13138-13145]. As part of an effort to elucidate the three-dimensional structure of the active complex formed between the labile drug and bulged DNA, we have studied the binding of stable drug products generated in the course of the cleavage reaction with oligodeoxynucleotides containing the bulged structure. By use of fluorescence quenching, we have found that one drug product, which is also formed in the absence of bulged DNA and most closely resembles the biradical intermediate in the cleavage reaction, specifically binds bulged DNA with a Kd in the low micromolar range and competitively inhibits the cleavage reaction. Other drug products, including one formed only in the presence of bulged DNA, fail to bind to the bulged DNA. Implications of these results for the proposed mechanism of bulge-specific cleavage and for the role of the DNA bulge in generating a unique drug product are discussed.

Structural basis for the sequence-specific DNA strand cleavage by the enediyne neocarzinostatin chromophore. Structure of the post-activated chromophore-DNA complex.

Neocarzinostatin chromophore (NCS chrom) belongs to a family of highly potent enediyne antitumor antibiotics which bind to specific DNA sequences and cause single- and/or double-strand lesions. NCS chrom-DNA complexes have eluded structural studies since the native form of the drug is extremely labile in aqueous conditions. We report the three-dimensional structure of the stable glutathione post-activated NCS chrom (NCSi-glu)-DNA complex [NCSi-glu-d(GGAGCGC).d(GCGCTCC)] using NMR and distance geometry-molecular dynamics simulation methods. NCSi-glu interacts with the GCTC tetranucleotide on one strand and with the AGC trinucleotide on the other strand through the unique intercalation at the 5'-CT/5'-AG step and minor groove binding. The DNA-drug complex exhibits an extended, unwound V-shaped intercalation site and wider and shallower grooves than the free DNA duplex. The structure of the complex manifests specific van der Waals interactions and H-bond formation between the carbohydrate moiety and a specific DNA sugar/phosphate. Prominent among those are the contacts of the NCSi-glu residues with the functional groups in the minor groove that are characteristic of individual DNA bases. These results provide a structural model for understanding the sequence specificity of the single- and double-strand cleavage at the AGC and related sites by the enediyne NCS chrom.

Spontaneous generation of a biradical species of neocarzinostatin chromophore: role in DNA bulge-specific cleavage.

Detailed structure determination of the major and minor base-catalyzed degradation products of the chromophore of the enediyne anticancer antibiotic neocarzinostatin in the absence of DNA demonstrates that the enolate Michael addition reaction leading to a spirolactone cumulene intermediate is a spontaneous, stereoselective process. The implications of these findings for the mechanism of the thiol-independent, site-specific cleavage by the so-generated radical species of the drug at a DNA bulge are described.