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.

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.

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.

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.

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.

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)

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.

Site-specific cleavage at a DNA bulge by neocarzinostatin chromophore via a novel mechanism.

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)

DNA conformation-induced activation of an enediyne for site-specific cleavage.

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.

Mismatch-induced switch of neocarzinostatin attack sites in the DNA minor groove.

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)

Neocarzinostatin acts as a sensitive probe of DNA microheterogeneity: switching of chemistry from C-1' to C-4' by a G.T mismatch 5' to the site of DNA damage.

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-induced hydrogen atom abstraction from C-4' and C-5' of the T residue at a d(GT) step in oligonucleotides: shuttling between deoxyribose attack sites based on isotope selection effects.

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').

Oxygen transfer from the nitro group of a nitroaromatic radiosensitizer to a DNA sugar damage product.

Mechanisms based on one-electron oxidation appear incomplete in explaining cellular radiosensitization by nitroaromatic compounds such as misonidazole. Evidence is presented for a novel mechanism that may be involved in enhancing DNA strand breakage due to a variety of agents, including ionizing radiation, that generate carbon-centered radicals on DNA deoxyribose. Under anaerobic conditions the carbon-centered radical generated selectively at C-5' of deoxyribose of thymidylate residues in DNA by the antitumor antibiotic neocarzinostatin reacts with misonidazole to produce a DNA damage product in the form of 3'-(formyl phosphate)-ended DNA. In an 18O-transfer experiment we find that the carbonyl oxygen of the activated formyl moiety (trapped as formyl-Tris) is derived from the nitro group oxygen of misonidazole. This result strongly supports a mechanism in which a nitroxide radical adduct, formed by the addition of misonidazole to the radical at C-5' of deoxyribose, cleaves between the N and O so as to form an oxy radical precursor of the formyl moiety and a two-electron reduction species of misonidazole.

Identification of 2-deoxyribonolactone at the site of neocarzinostatin-induced cytosine release in the sequence d(AGC).

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)

Atypical abasic sites generated by neocarzinostatin at sequence-specific cytidylate residues in oligodeoxynucleotides.

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.

3'-Formyl phosphate-ended DNA: high-energy intermediate in antibiotic-induced DNA sugar damage.

Under anaerobic conditions where the nitroaromatic radiation-sensitizer misonidazole substitutes for dioxygen, DNA strand breakage (gaps with phosphate residues at each end) by the nonprotein chromophore of the antitumor antibiotic neocarzinostatin (NCS-Chrom) is associated with the generation of a reactive form of formate from the C-5' of deoxyribose of thymidylate residues. Such lesions account for a minority (10-15%) of the strand breakage found in the aerobic reaction without misonidazole. Amino-containing nucleophiles such as tris(hydroxymethyl)aminomethane (Tris) and hydroxylamine act as acceptors for the activated formate. The amount of [3H]formyl hydroxamate produced from DNA labeled with [5'-3H]thymidine is comparable to the spontaneously released thymine. During the course of the reaction, misonidazole undergoes a DNA-dependent reduction and subsequent conjugation with glutathione used to activate NCS-Chrom. From these and earlier results, we propose a possible mechanism in which the carbon-centered radical formed at C-5' by hydrogen atom abstraction by thiol-activated NCS-Chrom reacts anaerobically with misonidazole to form a nitroxyl-radical-adduct intermediate, which fragments to produce an oxy radical at C-5'. beta-Fragmentation results in cleavage between C-5' and C-4' with the generation of 3'-formyl phosphate-ended DNA, a high-energy form of formate, which spontaneously hydrolyzes, releasing formate and creating a 3'-phosphate end, or transfers the formyl moiety to available nucleophiles. A similar mechanism, involving dioxygen addition, is probably responsible for the 10-15% DNA gap formation in the aerobic reaction.

Mechanism and base specificity of DNA Breakage in intact cells by neocarzinostatin.

When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase and treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO4 termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP much greater than CMP greater than GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanism of novel DNA sugar damage by an antitumour protein antibiotic.

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)

Activation of neocarzinostatin chromophore and formation of nascent DNA damage do not require molecular oxygen.

Thiol-activated neocarzinostatin chromophore abstracts tritium from the 5', but not from the 1' or 2' positions of deoxyribose in DNA and incorporates it into a stable, non-exchangeable form. The abstracted tritium remains covalently associated with the chromophore or its degradation product after treatment with acid or alkali, respectively. Drug activation and the consequent hydrogen abstraction reaction, presumably generating a carbon-centered radical at C-5', do not require molecular oxygen but have a dose-dependent relation with thiol. Under aerobic conditions, where base release and DNA strand breaks with nucleoside 5'-aldehyde at the 5'-ends are produced, hydrogen abstraction from C-5' parallels these parameters of DNA damage. It is possible to formulate a reaction scheme in which the carbon- centered radical at C-5' is an intermediate in the formation of the various DNA damage products found under both aerobic and anaerobic conditions.

Nitroaromatic radiation sensitizers substitute for oxygen in neocarzinostatin-induced DNA damage.

The ability of neocarzinostatin (NCS) chromophore to damage DNA, as manifested by strand breaks and base release, is markedly decreased under anaerobic conditions but can be restored by nitroaromatic radiation sensitizers, which by themselves have no effect. The effectiveness of these compounds is correlated with their electron affinity as measured by their one-electron reduction potentials and is inversely related to the concentration of thiol used to activate the NCS. Whereas strand breaks with thymidine 5'-aldehyde at the 5' end and released thymine are the main DNA damage products in O2, under anaerobic conditions misonidazole causes a marked increase in the release of thymine and in the formation of breaks with 5'- phosphate ends. In both cases the 3' end of the break carries a phosphate group, and the attack-site specificity of spontaneous and alkali-labile DNA strand breakage and base release are identical. In O2, misonidazole does not affect the extent of DNA damage or alter the distribution of DNA damage products found with NCS alone. The data do not distinguish whether the nitroaromatic compounds function by interacting with NCS-induced nascent damage on the DNA, by being converted by activated NCS into a DNA-damaging species, or by participating in the activation of NCS to a DNA-damaging species. The implications of these results for the treatment of hypoxic tumor cells with the combined use of radiomimetic drugs and radiation sensitizers are discussed.