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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.

[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.

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.

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.

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)

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.

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.

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.

The chromophore of the anticancer drug neocarzinostatin (NCS-Chrom) oxidatively cleaves single-stranded or duplex DNA site-specifically in the absence of activating thiol provided that the DNA contains a bulged structure. Point mutations, deletions, and insertions in the DNA analogue and its complement of the 3'-terminus of yeast tRNA(Phe) show that for a single-stranded DNA to be cleaved by NCS-Chrom the DNA must generate a hairpin structure with an apical loop and at least a two-base-pair stem hinged to a region of duplex structure via a bulge containing a target nucleotide at its 3' side. The size of the loop is not critical so long as it contains at least three nucleotides; the bulge requires a minimum of two nucleotides but must have fewer than five. With a notable exception involving base-pair changes immediately 3' to the bulge, base changes in the bulge and base-pair changes immediately 5' to the bulge retain substrate activity for NCS-Chrom. Maintenance of the bulged structure requires stable duplex regions on each side of the bulge. A similar bulged structure, but lacking a loop, formed by the annealing of a linear 8-mer and a 6-mer is an excellent target for cleavage in the thiol-independent reaction. Drugs such as netropsin, which sequester the DNA into nonbulge containing structures inhibit the reaction. In the absence of O2 strand cleavage is blocked and quantitatively replaced by a presumed drug-DNA covalent adduct.(ABSTRACT TRUNCATED AT 250 WORDS)

Neocarzinostatin chromophore (NCS chrom) was found to induce site-specific cleavage at the 3' side of a bulge in single-stranded DNA in the absence of thiol. This reaction involved the oxidative formation of a DNA fragment with a nucleoside 5'-aldehyde at its 5' terminus and generated an ultraviolet light-absorbing and fluorescent species of post-activated drug containing tritium abstracted from the carbon at the 5' position of the target nucleotide. The DNAs containing point mutations that disrupt the bulge were not cleavage substrates and did not generate this drug product. Thus, DNA is an active participant in its own destruction, and NCS chrom may be useful as a probe for bulged structures in nucleic acids.

Activation of the enediyne neocarzinostatin chromophore (NCS-Chrom) by thiol addition at C-12 generates a diradical species with radical centers at C-2 and C-6, which abstract hydrogens from deoxyribose in the minor groove of DNA. Since hydrogen abstraction from DNA accounts for only part of the hydrogen incorporated at these sites, it is important to determine the other possible sources. At low concentration of thiol, a condition resembling that during NCS-Chrom-induced DNA damage, the major non-DNA hydrogen donation source was found to be the carbon-bound hydrogen of the aqueous methanol solvent, rather than the expected sulfur-bound hydrogen of the thiol. Further, experiments with the gamma-L-glutamyl-DL-cysteinylglycine labeled with deuterium on the alpha- or beta-carbons to the sulfur showed small amounts of internal transfer of hydrogen into C-2 of the drug from the naturally occurring L,L diastereomer only. Quantitation of the hydrogen transfer was accomplished by separation of the L,DL diastereomeric mixtures of the thiol-NCS-Chrom adducts. In all, these various hydrogen donation sources can account for at least 70-80% of the hydrogen incorporated at C-2 of the drug under DNA damage conditions. Selective quenching of the radical at C-2 could account for the predominance of single-stranded over double-stranded DNA lesions.

Glutathione-activated neocarzinostatin chromophore (NCS-Chrom) generates bistranded lesions at AGC.GCT sequences in DNA, consisting of an abasic site at the C residue and a strand break at the T residue on the complementary strand, due to hydrogen atom abstraction from C-1' and C-5', respectively. Earlier work showed that 2H from C-5' of T was selectively abstracted by the radical center at C-6 of activated NCS-Chrom, supporting a proposed model of the active-drug/DNA complex. However, since under the conditions used breaks at the T exceeded their inclusion in bistranded lesions, it was not clear what fraction of the hydrogen transfer represented bistranded lesions. Since virtually all abasic sites at the C are part of a bistranded lesions, hydrogen transfer from C-1' of C into the drug should reflect only the bistranded reaction. Accordingly, a self-complementary oligodeoxynucleotide 5'-GCAGCICTGC-3' was synthesized in which the C contained 2H at the C-1' position. In order to eliminate an 2H isotope effect on the transfer and to increase the extent of the bistranded reaction, an I residue was substituted for the G opposite the C residue. Sequencing gel electrophoretic analysis revealed that under one-hit kinetics, 37% of the damage reaction was associated with abasic site (alkali-labile break) formation at the C residue and 48% with direct strand breaks at the T residue. Thus, 74% of the damage involved a bistranded lesion. 1H NMR spectroscopic analysis of the reacted chromophore showed that 2H had been selectively transferred into the C-2 position to the extent of approximately 22%.(ABSTRACT TRUNCATED AT 250 WORDS)

Based on the finding that the wobble G.T mismatch 5' to the C of AGC.GCT results in switching of the attack chemistry by neocarzinostatin chromophore (NCS-Chrom) on the deoxyribose moiety of C from C-1' to C-4' [Kappen, L. S. & Goldberg, I. H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6706-6710], a series of mismatches has been explored for their effect on the chemistry of damage at the T of AGT.ACT in oligodeoxynucleotides, a site at which 4'-chemistry ordinarily occurs. Placement of a G.T mispair 5' to the T results in a marked increase in 4'-chemistry, as measured by the formation of breaks with 3'-phosphoglycolate ends and abasic sites due to 4'-hydroxylation. Strikingly, 4'-chemistry is induced at the T on the complementary strand, a site ordinarily restricted to 5'-chemistry. Substitution of dioxygen by the radiation sensitizer misonidazole exerts a pronounced effect on the partitioning of the 4'-chemistry in favor of the 3'-phosphoglycolate product. Both stable T.G and unstable T.C mismatches at the attack site itself are associated with marked inhibition of damage at this site. Whereas placement of the relatively stable G.A mismatch on the 5'-side of the T residue (AGT) results in substantial inhibition of damage at the T without shifting of chemistry, the same mismatch at the 3'-side of the attack site decreases damage only slightly but is associated with the appearance of significant 1'-chemistry. By contrast, no shift in chemistry is found with bleomycin, which attacks at C-4'.(ABSTRACT TRUNCATED AT 250 WORDS)

The diradical form of thiol-activated neocarzinostatin chromophore resides in the minor groove of DNA, where it has access to hydrogen atoms at the C-5', C-1', and C-4' positions of deoxyribose on each strand. In a dodecamer oligodeoxyribonucleotide containing the sequence AGC.GCT, a bistranded lesion staggered two nucleotides in the 3' direction, is generated that consists primarily of an abasic site (2'-deoxyribonolactone) at the C due to 1' chemistry and a direct strand break at the T due to 5' chemistry. Sequencing-gel analysis reveals that 72% of the damage at the C results from 1' chemistry with minor lesions consisting of a strand break due to 5' chemistry (15%) and 4' chemistry (less than 2%) and an abasic site (4'-hydroxylation product) (12%) due to 4' chemistry. Replacement of the G.C base pair 5' to the C by a G.T wobble mismatch results in a remarkable switching of the chemistry of damage at the C from C-1' to C-4'. The 1' chemistry is almost eliminated and replaced by 4' chemistry, so that the latter accounts for 64% of the damage, mainly in the form of the 4'-hydroxylation product (abasic site) and a smaller amount of the DNA fragment with a phosphoglycolate at the 3' end (strand break). Substitution of the radiation sensitizer misonidazole for dioxygen markedly enhances partitioning of the 4' chemistry in favor of the glycolate-containing product. On the complementary strand the G.T mismatch results in an increase in 4' chemistry at the T residue, but 5' chemistry remains the main mechanism. When a G.A mismatch is inserted 5' to the C, there is a marked decrease in all damage at this site without detectable switching of chemistry. These results show that the diradical form of thiol-activated neocarzinostatin chromophore acts as sensitive probe of DNA microheterogeneity.

Neocarzinostatin (NCS) is an enediyne antitumor antibiotic that cleaves DNA following a thiol-induced electronic rearrangement to a diradical form. Structure-function studies with 11 thiol-containing compounds were undertaken to clarify the role of the thiol in NCS-mediated DNA damage. The rates of activation of NCS in the presence of DNA with the various thiols approximated a Brønsted relation (beta = 0.43, r2 = 0.86), which suggests that the basicity/nucleophilicity of the thiol is important to NCS activation. However, an additional contribution to NCS activation may arise from the affinity of the thiol for DNA, since there is a correlation between the concentration of thiol producing maximal DNA damage, assessed by quantitating the topologic forms of plasmid pBR322 following treatment with NCS, and the apparent ability of the thiol to bind to DNA by hydrophobic or electrostatic interactions. The overall second-order rate constants for the activation of NCS were found to be inversely correlated with the thiol optima; a plot of the former versus the reciprocal of the optimal thiol concentration revealed a first-order rate constant of activation of 0.013 s-1 in the presence of DNA. This indicates that maximal DNA damage occurs when NCS is activated with a half-life of 52 s, a relatively slow rate of activation that suggests that NCS binds to DNA before undergoing activation by thiol. Finally, an analysis of strand breaks in pBR322 shows that thiols possessing a carboxylate moiety produce larger quantities of bistranded DNA lesions than their esterified or non-carboxylate-containing counterparts.

Double-strand (DS) DNA damage caused by neocarzinostatin (NCS) has been studied in the trinucleotide AGT-ACT sequence in an AP-1 transcription factor binding site. There are strong similarities between bistranded lesions produced at AGT.ACT and AGC-GCT, including the fact that DS lesions outnumber SS lesions on the AGT and AGC strands, while SS exceed DS on the ACT and GCT strands. Structure-function studies revealed that a variety of different thiols produced bistranded lesions in this model by predominantly C4'-hydrogen atom abstraction (84-93%) at the T of AGT and C5'-hydrogen atom abstraction (87-91%) at the T of ACT. Single-strand (SS) lesions were found to represent a variable mixture of C4' and C5' chemistry. The C4'-hydroxylated abasic site occurred in both SS and DS lesions at both sites and accounted for most of the DS damage at AGT (60-83%); the remaining damage consisted of 3'-phosphoglycolate- and 3'-phosphate-ended fragments. The nature of the thiol was found to affect the partitioning of the breakdown products arising from C4' and, to a lesser extent, C5' hydrogen atom abstraction. Production of 3'-phosphoglycolate residues, restricted mainly to the T of AGT in bistranded lesions, correlated with the incidence of direct DS breaks in the AGT.ACT model and in plasmid DNA and appeared to be influenced by the reducing power of the thiol activator. Furthermore, hydrazine and sodium borohydride both inhibited the formation of glycolate, an effect that was exploited to determine the rate constant for 3'-phosphoglycolate formation: 0.06 min-1 at 0 degree C, pH 7.4. Under anaerobic conditions, the nitroaromatic radiation sensitizer misonidazole caused a large increase in glycolate production in both SS and DS lesions formed by NCS, which suggests that the formation of 3'-phosphoglycolate, like 3'-formylphosphate generated by C5' chemistry, involves an oxyradical intermediate. The pathways for DNA damage involving C4' and C5' hydrogen atom abstraction thus share many common features, several of which are consistent with a mechanism for the production of NCS-mediated bistranded lesions at AGT.ACT that involves a tetraoxide bridge joining the lesions on opposite strands of DNA.

Use has been made of the mechanism of DNA deoxyribose damage by the ene-diyne-containing chromophore of the antitumor antibiotic neocarzinostatin to provide chemical evidence for the structure of the activated drug-DNA complex. Radical centers at C-2 and C-6 of the diradical form of the glutathione-activated chromophore abstract hydrogen atoms from C-1' of the C residue and C-5' of the T residue in AGC.GCT to generate a bistranded lesion consisting of an abasic site at C and a strand break at T. This laboratory has proposed a molecular model for the drug-DNA interaction in which the naphthoate moiety of the chromophore intercalates between A.T and G.C, placing the diradical core in the minor groove, so that the radical centers at C-6 and C-2 are close to C-5' of T and C-1' of C, respectively. To determine which radical center abstracts one of the hydrogen atoms from C-5', the self-complementary oligodeoxynucleotide GCAGCGCTGC was synthesized with 2H at both 5' positions of the T residue and treated with glutathione-activated chromophore. Sequencing-gel electrophoresis showed that drug attack was limited to the T and C residues and that abstraction of 2H from C-5' exhibited a small isotope selection effect of 1.25. 1H NMR spectroscopic examination of the reacted chromophore, isolated by HPLC, indicated that 2H was selectively abstracted by C-6, providing experimental corroboration of the model and further elucidating the chemical mechanism. Since direct strand breakage at the T residue exceeds (44% more) abasic site formation at the C residue, other models of drug-DNA interaction leading to only single-strand breaks are also considered.

The thiol-activated neocarzinostatin chromophore cleaves duplex oligonucleotides containing the sequence-TGTTTGA-, producing 3'-phosphoglycolate and 3'-phosphate fragments at T, indicating the involvement of 4'- as well as 5'-chemistry at this residue. Substitution of deuterium for hydrogen at the C-4' position of the affected T leads to a kinetic isotope effect (kH/kD) of 4.0 on the formation of the glycolate-ended product, whereas deuterium at C-5' of the same T reveals kH/kD of 1.6 in the formation of the phosphate-ended product. The proportion of the products representing 4'- and 5'-chemistry can be shifted on the basis of isotope selection effects. A second product resulting from 4'-chemistry, the abasic site associated with 4'-hydroxylation, has been identified as an alkali-labile site, and as a pyridazine derivative formed after cleavage by hydrazine. A comparable isotope effect on its production (kH/kD = 3.7) relative to that of 3'-phosphoglycolate production is consistent with a common intermediate, a putative 4'-peroxy radical, in their formation. The formation of both products of 4'-chemistry is oxygen-dependent, and the internal partitioning between them (3'-phosphate or 3'-phosphoglycolate) is influenced by thiols. Moreover, the nitroaromatic radiation sensitizer misonidazole can substitute for dioxygen, yielding 3'-phosphoglycolate and alkali-labile 3'-phosphate ends, indicative of 4'-chemistry. In addition to the internal partitioning of 4'-chemistry, thiols also affect the overall extent of cleavage (4' plus 5') and the relative partitioning between both sites of attack (4' or 5').

Direct double-strand breaks in DNA have been implicated in cellular lethality of the antitumor antibiotic neocarzinostatin, but the mechanism of their formation has not been elucidated. Evidence is presented that neocarzinostatin causes sequence-specific direct double-strand breaks whose formation is strongly influenced by the activating thiol. Seven-fold more double-strand breaks result when glutathione rather than 2-mercaptoethanol is used to activate the drug to its putative diradical form, while the sequence specificity of cleavage remains the same. These data explain earlier inconsistencies in the ratios of double-strand to single-strand breaks obtained from in vitro and in vivo studies. Double-strand cleavage sites, occurring predominantly at GT steps, especially AGT.ACT, consist of trinucleotide sequences with a two-nucleotide 3'-stagger of the cleaved residues. The chemical structures of the cleavage sites suggest a model in which a neocarzinostatin-induced double-strand break results from abstraction of a C5' hydrogen atom from the T of ACT and the C4' hydrogen atom of the T of AGT by a single molecule of the diradical form of the drug. Single-strand breaks at these sites occur as separate events with attack at the C5' hydrogens. These findings permit the generalization that single-strand breaks produced by neocarzinostatin show a base preference but no clear sequence specificity, while bistranded lesions are sequence-specific in nature.

Model building and molecular mechanics and dynamics calculations have been performed on a number of complexes of the post-activated form of the neocarzinostatin chromophore (NCS) with the B-DNA oligomer 5'GAGCG:5'CGCTC. Stable structures with the naphthoic acid moiety intercalated at all base pairs can be constructed. The observed bistranded lesions consisting of an abasic site at the Cyt residue in AGC and a direct break at the Thy residue on the complementary strand can be explained by assuming that NCS in the (R,R) form intercalates between the Ade2-Thy9/Gua3-Cyt8 base step with its 'diradical' core oriented towards the 3'-end of the (+) strand. Sites at C5', C4' and C1' in the minor groove are within a short enough distance from the two radical centers on NCS to permit hydrogen atom abstraction and the formation of the bistranded lesions. Strand cleavage at Thy9 may occur as a single lesion if NCS is intercalated into the Gua3-Cyt8/Cyt4-Gua7 base step with its active core towards the 3'-end of the (-) strand. The results are analyzed, and the utility and limitations of this type of model building are discussed.

Neocarzinostatin (NCS), an antitumor protein antibiotic that causes strand scissions of DNA both in vitro and in vivo, is shown to lower the template activity of DNA for DNA polymerase Iin vitro. There is a correlation between the extent of strand scission and the degree of inhibition, maximal inhibition of the polymerase reaction being obtained under conditions promoting maximal strand scission. These effects can be related to the concentrations of NCS and of 2-mercaptoethanol and are maximized by pretreatment of the DNA with drug. Results from polymerase assays in which the amount of drug-treated DNA template was varied at a constant level of the enzyme suggest that the sites associated with NCS-induced breaks are nonfunctional in DNA synthesis but bind DNA polymerase I. The binding of the enzyme to the inactive sites is further confirmed using [203 Hg] polymerase. It is shown that the lowering of the template activity of DNA by NCS under conditions of strand scission is due to the generation of a large number of inactive sites that block, competitively, the binding of DNA polymerase to the active sites on the template. Furthermore, the inhibition of DNA synthesis, which depends on the extent of strand breakage and on the relative amounts of template and enzyme, can be reversed by increasing the levels of template or polymerase. The finding that DNA synthesis directed by poly [d(A-T)] is much more sensitive to NCS than that primed by poly [d(G-C)] suggests that the drug preferentially interacts at regions containing adenine and/or thymine residues.

When the nonprotein chromophore of neocarzinostatin was allowed to react with either calf thymus DNA or poly(dA-dT) . poly(dA-dT) in the presence of 2-mercaptoethanol and the DNA was precipitated with ethanol, 5% of the fluorescence attributable to the naphthalene rings of the chromophore coprecipitated with the DNA. Most of this fluorescence remained attached to DNA through successive reprecipitations, suggesting formation of covalent adducts between chromophore and DNA. Enzymatically digested poly(dA-dT) . poly(dA-dT)-chromophore adduct contained, in addition to deoxyadenosine and thymidine, several highly fluorescent hydrophobic products, separable by reverse-phase chromatography, all of which contained both adenine and thymine radiolabel, as well as chromophore radiolabel. One such product consistently had twice as much thymine as adenine, suggesting a structure chromophore-d(TpApT), in which the attached chromophore rendered both phosphodiester bonds refractory to endonuclease S1. This adduct fragment was completely hydrolyzed at pH 12, releasing adenine, 3'-dTMP, and 5'-dTMP. At pH 7, the adduct fragment slowly released chromophore and 3'-dTMP with parallel kinetics, leaving a modified d(ApT), which was cleaved by snake venom phosphodiesterase to yield 5'-dTMP and a modified deoxyadenosine. These hydrolysis patterns are unlike those of any previously characterized base or phosphotriester DNA adduct but rather indicate an altered deoxyadenosine sugar. The formation of adducts containing a modified deoxyribose suggests that deoxyribose may be the site of covalent chromophore attachment. Alteration of this same site, possibly the 5'-carbon of the sugar moiety, may account for the extreme lability of the phosphodiester bond.

Neocarzinostatin- (NCS) induced release of cytosine from the deoxycytidylate residues of d(AGC) sequences of duplex oligonucleotides leaves a damaged sugar residue with intact phosphodiester linkages [Kappen, L.S., Chen, C., & Goldberg, I.H. (1988) Biochemistry 27, 4331-4340]. In order to isolate and characterize the sugar damage product, drug-treated duplex d(AGCGAGC*G) (the single target C* residue has 3H in its 5- and 5'-positions) was enzymatically digested to mononucleosides. High-pressure liquid chromatographic analysis of the digest revealed drug-induced products which could be cleanly separated by thin-layer chromatography (TLC) into two components: product a (Rf0.47) and product 1 (Rf0.87). The more polar product a was further purified by adsorption onto DEAE-Sephadex A-25. After elution with HCl and lyophilization, this material behaved like product 1 on TLC. Readjustment to alkaline pH caused its quantitative conversion back to product a. On electrophoresis product 1 behaved like a neutral compound, and the negatively charged product a migrated just behind formate. On the basis of the various chemical and biochemical characteristics of the lesion and apparent 3H abstraction by NCS from the C-1' position, it appears that the two interconvertible products a and 1 are respectively the acid (carboxylate) and lactone forms of 2-deoxyribonic acid. The structure of the sugar damage product was confirmed by gas chromatography/mass spectrometry. The amount of 2-deoxyribonolactone recovered is about 60% of the cytosine released on a molar basis, showing that it is the major, if not the only, product associated with cytosine release.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Treatment of CHO cells with low doses of the protein antibiotic neocarzinostatin severely inhibited DNA replicon initiation but had no effect on chain elongation. The selectivity of the effect on initiation, which was greater than that seen with other chemical agents and comparable to that seen with X-rays, explains the biphasic dose response seen for DNA synthesis inhibition by this drug. Parallel experiments employing the nucleoid sedimentation technique indicated that half-maximal relaxation of domains of DNA supercoiling and half-maximal inhibition of replicon initiation required the same dose of neocarzinostatin, approximately 0.03 micrograms/mL. These results, similar results obtained with the protein antibiotic auromomycin, and previous results obtained with X-rays suggest a quantitative correlation between inhibition of replicon initiation and induction of sufficient strand breakage to relax domains of supercoiling in DNA of mammalian cells. Results in human ataxia telangiectasia fibroblasts indicated that neocarzinostatin, like X-rays, is much less effective in inhibiting DNA synthesis in these cells than in normal human fibroblasts. This finding is consistent with the hypothesis that the genetic defect in ataxia telangiectasia involves a failure to recognize the presence of strand breaks in cellular DNA.

Among the lesions induced in DNA by neocarzinostatin chromophore are spontaneous and alkali-dependent base release, sugar damage, and single-strand breaks with phosphate (PO4) at their 3' ends and PO4 or nucleoside 5'-aldehyde at the 5' ends. By measuring alkali-dependent thymine release and decomposition of the 5'-terminal thymidine 5'-aldehyde in drug-cut DNA, we show that the kinetics are the same for each process and that the nucleoside aldehyde is the source of about 85% of alkali-dependent thymine release. Reduction of the 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase permits their selective quantitation. Nucleoside 5'-aldehyde so measured accounts for over 80% of the drug-generated 5' ends; the remainder have PO4 termini. Since these techniques also include the contribution of alkali-labile sites in the measurement of PO4 ends, DNA sequencing was used to measure the ends directly. Using 3'-32P end-labeled DNA restriction fragments as substrates for the drug, it was found that drug attack at a T results in mainly two bands--the stronger one represents oligonucleotide with 5'-terminal nucleoside 5'-aldehyde and may account for over 90% of a particular break. Its structure was verified by its isolation from the sequencing gel, followed by various chemical and enzymatic treatments. In each case, the mobility of the product on the gel was altered in a predictable manner. In addition to spontaneous breaks, neocarzinostatin also causes alkali-labile breaks preferentially at T residues. These sites are heterogeneous in their sensitivity to alkali and are protected by reduction.

The nonprotein chromophore of neocarzinostatin forms a variety of adducts with DNA. The predominant adduct recovered from nuclease digests of chromophore-treated poly(dA-dT). poly(dA-dT) is a compound with structure chromophore-d(TpApT). Mild acid hydrolysis of this compound released free adenine, while snake venom exonuclease (pH 6.5) released 5'-dTMP leaving in both cases adducts of slightly altered chromatographic mobility. These results eliminate adenine and 5'-dTMP as possible sites of covalent chromophore attachment. Electrophoresis data suggest that the adduct is not a phosphotriester. At pH 8.6, chromophore-d(TpApT) spontaneously hydrolyzed, releasing chromophore and 3'-dTMP, leaving a modified d(ApT) which contained deoxyadenosine-5'-aldehyde. Deoxyadenosine-5'-aldehyde was released from the modified d(ApT) by snake venom exonuclease, and identified by a series of derivatizations including 1) mild oxidation to deoxyadenosine-5'-carboxylic acid, 2) NaBH4 reduction to deoxyadenosine, and 3) formation of a hydrazone with phenylhydrazine. Since deoxyadenosine-5'-aldehyde cannot exist as such in the chromophore-d(TpApT) adduct, we suggest that the chromophore may be covalently attached to the C-5' of deoxyadenosine as a phosphorylacetal or similar structure. Hydrolysis of the chromophore-acetal bond at pH 8.6 would leave a phosphorylhemiacetal on C-5', which would be expected to spontaneously decompose to yield the observed 3'-phosphate and 5'-aldehyde groups.

Neocarzinostatin chromophore produces alkali-labile, abasic sites at cytidylate residues in AGC sequences in oligonucleotides in their duplex form. Glutathione is the preferred thiol activator of the drug in the formation of these lesions. The phosphodiester linkages on each side of the abasic site are intact, but when treated with alkali, breaks are formed with phosphate moieties at each end. Similar properties are exhibited by the abasic lesions produced at the purine residue to which the C in AGC is base-paired on the complementary strand. The abasic sites at C residues differ from those produced by acid-induced depurination in the much greater lability of the phosphodiester linkages on both sides of the deoxyribose, in the inability of NaBH4 to prevent alkali-induced cleavage, and in the relative resistance to apurinic/apyrimidinic endonucleases. The importance of DNA microstructure in determining attack site specificity in abasic site formation at C residues is shown not only by the requirement for the sequence AGC but also by the findings that substitution of G by I 5' to the C decreases the attack at C, whereas placement of an I opposite the C markedly enhances the reaction. Quantitation of the abstraction of 3H into the drug from C residues in AGC specifically labeled in the deoxyribose at C-5' or C-1',2' suggests that, in contrast to the attack at C-5' in the induction of direct strand breaks at T residues, abasic site formation at C residues may involve attack at C-1'. Each type of lesion may exist on the complementary strands of the same DNA molecule, forming a double-stranded lesion.

Neocarzionstatin (NCS)-induced strand breakage of DNA generates nonfunctional binding sites for the E. coli DNA polymerase I. Treatment of the NCS-nicked DNA with alkaline phosphatase at 65 degrees C prior to the polymerase reaction results in 60-100-fold stimulation of dTMP incorporation whereas in a control not treated with the drug there is only a 2-fold increase. Sites of strand scission on the NCS-treated DNA bear phosphate at the 3' termini. This conclusion is supported by the kinetics of release of inorganic phosphate from NCS-cut DNA by exonuclease III. Since our earlier work has shown that virtually all the 5' ends of the nicks caused by NCS bear phosphomonoester groupings, the 3'- and 5'- phosphoryl termini could be quantitated using alkaline phosphatase and exonuclease III. Over a wide range of drug levels the amount of inorganic phosphate released by alkaline phosphatase is approximately twice as much as that removed by exonuclease III, indicating the presence of equal amounts of 3'- and 5'- phosphoryl termini. This, taken together with other previously demonstrated effects of NCS on DNA, such as the introduction of nicks not sealable by polynucleotide ligase, the release of thymine, and the formation of a malonaldehyde type compound, suggests that NCS-induced strand breakage involves base release accompanied by opening of the sugar ring with destruction of one or more nucleosides and results in a gap bounded by 3'- and 5'- phosphoryl termini.

The site responsible for the mercaptan (or borohydride)-stimulated DNA scission activity of neocarzinostatin chromophore (NCS-Chrom) is located in the central C12-subunit of the molecule. This has been determined by studies of the characteristic spectral properties of the chromophore and its reduction products and of the spectral changes induced by their interaction with its apoprotein (apo-NCS) and DNA. The UV-visible absorption, fluorescence, CD, and MCD spectral properties of the major nonprotein chromophoric component of neocarzinostatin (NCS-Chrom A) are assigned to its component substructures, the 2-hydroxy-5-methoxy-7-methyl-1-naphthoate, the five-membered cyclic carbonate ring (1,3-dioxolan-2-one), the 2,6-dideoxy-2-methylaminogalactose, and the incompletely defined C12-subunit which links the other three residues. Although the major source of its UV-visible absorption is the naphthoic acid residue (HNA-NCS), a significant absorption from approximately 260 to 330 nm is due to the presence of the highly unsaturated C12-subunit. The presence of the C12-subunit and, to a lesser extent, the cyclic carbonate reduces the intensity of the fluorescence emission of the fluorophore of NCS-Chrom A, the HAN-NCS subunit. The CD activity of NCS-Chrom A is also due to the presence of the C12-subunit. The MCD activity of NCS-Chrom A, however, is completely accounted accounted for by the naphthoic acid residue. A role for the C12-subunit in the binding of NCS-Chrom to DNA or apo-NCS is indicated by the resultant absorption hypochromicity in the region assigned to the C12-moiety. Limited modification of the C12-subunit (by mercaptan or borohydride) inactivates the chromophore for DNA strand scission, although both products still bind DNA. The naphthoic acid residue alone is not sufficient for binding to DNA. Therefore, the intercalation of the naphthoate residue between DNA base pairs requires the binding of NCS-Chrom to DNA probably via electrostatic interaction between the positively charged 2-methylamino group of the galactose residue and the negatively charged oxygens of the phosphate in the DNA backbone. The C12-unit is viewed as forming a short-lived reduction-activated species that, in the presence of oxygen, causes single-strand breaks in DNA. The cyclic carbonate residue is not required for in vitro DNA strand scission activity but affects its stability with respect to hydrolysis and reactivity with mercaptan. If DNA is absent the activated species decompose to inactive products, including a mercaptan (or hydrogen) addition product of the C12-subunit.

The reversible binding of neocarzinostatin chromophore to polynucleotides was studied in order to understand the molecular basis of its base sequence-specificity in DNA damage production. Studies of the spectroscopic and thermodynamic properties of chromophore-polynucleotide interactions reveal that the binding of the chromophore to poly(dA-dT) is qualitatively and quantitatively different from that to poly(dG-dC) (and poly(dI-dC]. From these and other experiments using double-stranded mixtures of homopolynucleotides, it is proposed that the observed A T specific intercalation might result from differential recognition of minor variations in the B-DNA type structure by the chromophore.

The antitumor antibiotic neocarzinostatin exhibits its main drug action by abstracting hydrogen from DNA deoxyribose with consequent strand breakage or related lesions. All biological activities of the drug derive solely from a nonprotein chromophoric substance (NCS-chrom) consisting of a novel epoxy-bicyclo-diyne-ene system. Thiol or sodium borohydride activates NCS-chrom into a labile, reactive species that induces DNA damage but causes inactivation of the drug in the absence of the target DNA. Mass spectrometric studies indicate that the isolated thiol-activated NCS-chrom product in the presence of DNA has the same molecular weight as the thiol-inactivated NCS-chrom product in the absence of DNA. No deuterium is incorporated into the chromophore from the deuterium-labeled sulfhydryl group. Since three deuterium atoms can be incorporated into the drug by treatment with sodium borodeuteride without DNA, adding an unlabeled DNA under parallel conditions permitted the ready identification of the activated NCS-chrom product that abstracted hydrogen from the DNA. Not only does the activated NCS-chrom product have the same structure as the inactivated drug without DNA, but two of the incorporated deuterium atoms have been substituted by hydrogen. With the aid of NMR spectrometry, the two replaced hydrogen atoms are found to be incorporated into the C-2 and C-6 positions of the bicyclo-diyne-ene ring of NCS-chrom and are derived neither from borodeuteride nor from the hydroxyl functions of the solvents. In accord with current proposals, the two hydrogens incorporated into the drug may come from closely opposed sites on the complementary strands of the DNA at which the drug is bound.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectroscopic analysis of the reduction of both nitro blue tetrazolium and ferricytochrome c induced by neocarzinostatin shows that superoxide free radical is produced during the spontaneous degradation of the antibiotic. The amount of superoxide free radical produced from neocarzinostatin is not affected by the presence of thiol, although earlier work has shown that DNA damage is stimulated at least 1000-fold by thiol. Transition metals are not involved in this reaction. Although superoxide dismutase inhibits the reduction of nitro blue tetrazolium and cytochrome c induced by neocarzinostatin, neither it nor catalase interferes with the action of neocarzinostatin on DNA, whether or not drug has been activated by thiol. The pH profiles for spontaneous base release and alkali-labile base release (a measure of nucleoside 5'-aldehyde formation at a strand break) do not correspond with that for the generation of superoxide free radical from neocarzinostatin. The same holds for supercoiled DNA cutting by neocarzinostatin chromophore in the absence of a thiol, which is an acid-favored reaction. These results indicate that the generation of superoxide free radical by the drug does not correlate with DNA damage activity, whether or not thiol is present. Furthermore, the failure of hydroxyl free-radical scavengers to inhibit drug-induced single-strand breaks in supercoiled DNA in the absence of thiol also indicates that a diffusible hydroxyl free radical is most probably not involved in this reaction.

The nucleotide sequence specificity of neocarzinostatin (NCS), auromomycin (AUR), bleomycin (Blm), phleomycin (Phlm), and tallysomycin (Tlm) has been determined by using these antibiotics and their associated chromophores to create strand scissions in end-labeled restriction fragments of DNA and then determining the base sequence of the oligonucleotides formed. NCS and the NCS chromophore induce similar patterns of cleavage in DNA fragments labeled at the 5' terminus. The pattern produced by the AUR chromophore also resembles that of its holoantibiotic. Dithiothreitol enhances the rate of cleavage of DNA by the AUR chromophore but does not alter the sequence specificity. The results suggest that the polypeptide component of AUR and NCS serves primarily as a carrier for the chromophore. When tested with a fragment labeled at the 3' terminus, the products of NCS and AUR cleavage do not display the patterns of chemically produced oligonucleotides cleaved at phosphodiester bonds, suggesting that the 5' terminus is modified by a sugar fragment. NCS primarily attacks thymine (75% of the total bases attacked) and, to a lesser extent, adenine (19%) and cytosine (6%). AUR preferentially attacks guanine (67% of total bases), while attacking less often thymine (24%) and adenine (9%). Bleomycin and its analogues preferentially cleave purine--pyrimidine (5' leads to 3') and pyrimidine--pyrimidine (3' leads to 5') sequences. All (5' leads to 3') GT and GC sequences were cleaved. Phlm G and Phlm-Pep are less active than bleomycin toward purines while Tlm was more active. The patterns of cleavage produced by Blm A2 and Blm B6 are similar, while those produced by Phlm-Pep, Phlm G, Blm-B1', and Blm-Pep resemble one another. The cleavage pattern of Tlm shows quantitative differences from the other analogues tested. Differences between bleomycin and its analogues may be related to structural differences in these molecules.

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.

Reaction of the antitumor protein neocarzinostatin with 1,2-cyclohexanedione in 0.25 M borate buffer, pH 9.0, resulted in complete modification of arginine residues in positions 66, 67, and 78. The arginine-modified protein lost its native structure and was biologically inactive in the inhibition of growth of HeLa cells, inhibition of DNA synthesis, and in vitro DNA strand scissions. Trypsin hydrolysis of 1,2-cyclohexanedione-modified neocarzinostatin resulted in selective cleavage of the Lys-Val (positions 20 and 21) bond of the primary structure yielding NH2-terminal 1-20 and the COOH-terminal 21-109 residue fragments. The latter contained modified arginine residues. Both peptide fragments were biologically inactive. Treatment of the arginine-modified neocarzinostatin and the arginine-protected 89-residue fragment with 0.25 M Tris-acetate buffer, pH 9.0, for 15 h resulted in the release of 1,2-cyclohexanedione, regenerating all three arginine residues. The regenerated protein and the 89-residue fragment were fully active biologically. Further, the regenerated 89-residue fragment possessed 70% of the reactivity of neocarzinostatin with antibody raised against the native protein. The conformation of the 89-residue fragment was almost identical with that of the native protein in CD spectral properties.

The structures of mercaptan and sodium borohydride reaction products of neocarzinostatin chromophore A (NCS Chrom A) are compared. Implications on the mechanism of activation of NCS are discussed.

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.

To elucidate the mechanism of the cell killing activity of neocarzinostatin on mammalian cells, the drug-induced damage of DNA and its repair were examined. Very low doses of neocarzinostatin, at which high survival of cells was observed, clearly produced single-strand breaks of DNA and decomposition of the 'DNA complex', but these damages appeared to be repaired almost completely. At higher doses of neocarzinostatin, single-strand breaks were repaired to a considerable extent while double-strand breaks seemed not to be repaired. The number of non-repairable single-strand breaks was about twice that of double-strand breaks. This implies that single-strand breaks are repaired except for those constituting double-strand breaks. Although at low levels of neocarzinostatin repair of double-strand breaks may occur, the correlation existing between the colony-forming ability of cells treated with neocarzinostatin and non-repairable DNA breakage suggests that production of a small number of critical non-repairable double-strand breaks per cell may be responsible for the cell killing activity of the drug.

The microstructural requirements for optimal interaction of neocarzinostatin chromophore (NCS-C) with DNA have been investigated using a series of hexadeoxyribonucleotides with modified bases such as O6-methyl G (MeG), I, 5-methyl C (MeC), U, or 5-Bromo U (BrU) at specific sites in its preferred trinucleotide 5'GNaNb3':5'Na,Nb,C3' (Na = A, C, or T). Results show that MeG:C and G:MeC in place of G:C improve direct strand cleavage at the target Nb (Nb = T greater than A much greater than C greater than G), whereas MeC:G and C:MeG in place of Na:Nb, hinder cleavage. The optimal base target at Nb appears to be determined by its ability to form T:A type base pairing instead of C:G type. The observed differences in DNA strand cleavage patterns can be rationalized by induced changes in target site structure and are compatible with a model for NCS-C:DNA interaction in which the naphthoate moiety intercalates between 5'GNa3', and the activated tetrahydro-s-indacene, lying in the minor groove, abstracts a hydrogen atom from C-5' of Nb.

Neocarzinostatin, a protein with antibiotic activity, is a bacterial mutagen. We have investigated the mutagenicity of neocarzinostatin towards Salmonella typhimurium and discovered that, unlike the situation in Escherichia coli, neocarzinostatin will revert base pair substitution mutations (missense or nonsense). However, when the R46 factor derivative, plasmid pKM101, was introduced, the mutagenicity of neocarzinostatin towards base pair substitution-carrying mutants of S. typhimurium was readily detected. Neocarzinostatin had only modest activity in reverting a frameshift mutation in S. typhimurium, but that activity, too, required the presence of pKM101. Mutant pKM101 plasmids which no longer enhanced mutagenesis also lost their ability to promote neocarzinostatin-induced mutations. Finally, the umuC36 mutation, which renders E. coli nonmutable by ultraviolet light, also rendered the bacteria nonmutable by neocarzinostatin. The effect of the umuC36 mutation was suppressed by plasmid pKM101.

Neocarzinostatin (NCS) belongs to a family of antitumour protein antibiotics that selectively inhibit DNA synthesis. Replicon initiation in mammalian cells is selectively inhibited by NCS, and cells defective in DNA repair, such as ataxia telangiectasia fibroblasts, are especially sensitive to NCS as they are to X-ray. The holoantibiotic consists of a nonprotein chromophore (Mr = 659), tightly and specifically bound to an apoprotein (Mr = 10,700). The apoprotein protects the highly labile chromophore from degradation in aqueous solution; all the activity resides in the nonprotein chromophore. The latter binds specifically to DNA, especially to regions rich in T and A residues, with a tight binding site consisting of four base pairs. NCS chromophore consists of three main structural subunits: a naphthoic acid derivative, an amino-sugar and a connecting highly unsaturated middle component (C12H5) with a strained ether (probably epoxide) and cyclic carbonate. The authors have proposed that the naphthoic acid subunit intercalates DNA and the positively charged amino sugar binds electrostatically to the negatively charged sugar phosphate backbone of DNA; these two anchors serve to juxtapose the middle piece with the deoxyribose of mainly thymidylate residues in DNA. Upon activation of the drug by a thiol (which forms an adduct with the middle piece) and in the presence of O2, there is a selective oxidation of the 5'-C of deoxyribose to produce a DNA strand break with a phosphate at the 3'-end and a nucleoside 5'-aldehyde at the other. Kinetic analysis shows that one molecule of thiol adds to DNA-bound NCS chromophore even in the absence of oxygen; this is rapidly followed by the consumption of 1 mol of O2 and then another mol of thiol. The oxygen of the 5'-aldehyde is derived from O2, not H2O. Even in the absence of O2 the NCS chromophore abstracts a hydrogen from C-5' of deoxyribose in DNA, presumably generating a carbon-centred radical intermediate in the DNA (other mechanisms have not been eliminated) which can add O2 to form a peroxy derivative. The second molecule of thiol may be involved in the cleavage of this complex to form the 5'-aldehyde at the strand break. There is no evidence for the involvement of metals or a diffusible form of reduced oxygen.(ABSTRACT TRUNCATED AT 400 WORDS)

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C12-subunit of the previously determined partial structure 2a (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom A) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C12-substructure, whereas a parallel two-step reduction occurs on NaBH4 treatment. Both reactions occur with rearrangement of the C12-substructure and the implication for the mechanism of action of NCS-Chrom A in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom A as well as minor components B and C by two protons.

The anti-tumor antibiotics neocarzinostatin and bleomycin specifically oxidize deoxyribose in DNA at the C-5' and C-4' positions, respectively. The resulting DNA lesions include strand breaks and apyrimidinic sites. Both agents are broad specificity mutagens, inducing, in various systems, base substitutions, frameshifts and deletions. Sequencing studies in bacterial systems have suggested that the base substitutions may result primarily from replicative bypass of the oxidized apyrimidinic sites.

The sequence-specific interaction of neocarzinostatin chromophore (NCS-C) has been evaluated with a series of synthetic oligodeoxyribonucleotides of defined sequences containing the most preferred nucleotide cleavage site, T or A, or both. NCS-C preferentially cleaves T or A residues in the sequence GN1N2, where N2 is T or A. Greater cleavage occurs on the strand enriched with G residues, provided that they are adjacent to other G residues, but not at N1. These results are compatible with a model for drug binding in which the naphthoate moiety of NCS-C preferentially intercalates at GN1. This is accompanied by electrostatic binding interaction provided by the positively charged amino sugar moiety so as to place the reactive bicyclo[7.3.0]dodecadienediyne epoxide moiety in an appropriate orientation in the minor groove enabling, upon thiol activation, attack at C-5' of T or A. At certain sequences, such as GCT.AGC, a similar binding mode is also able to generate a basic lesions at the C residue on the opposite strand, forming a bistranded lesion. Although the reactions with glutathione generally show the same strand selectivity and sequence specificity as those with dithiothreitol, the former is usually more efficient than the latter.