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.

Neocarzinostatin induction of DNA repair synthesis in HeLa cells and isolated nuclei.

The antitumor antibiotic neocarzinostatin that causes DNA strand breaks in vivo and in vitro is shown to induce DNA repair synthesis in HeLa S3 cells. In the repair assay, the parental DNA was prelabeled with 32P and a density label (bromodeoxyuridine) was introduced into the new synthesized DNA. Quantitation of the repair synthesis as measured by the incorporation of [3H]thymidine into the light parental DNA at varying doses of the drug indicate that there is a significant repair response at low levels of the drug (0.2--0.5 microgram/ml) which cause DNA strand breakage and inhibition of DNA synthesis. In isolated HeLa nuclei neocarzinostatin stimulates the incorporation of dTMP many-fold. This enhancement of dTMP incorporation, which requires the presence of a sulfhydryl agent, is a consequence of the drug-induced DNA strand breakage and is in the parental DNA. These results suggest that an intact cell membrane is not required for DNA strand breakage and its subsequent repair.

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)

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)

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)

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.

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)

Deoxyribonucleic acid damage by neocarzinostatin chromophore: strand breaks generated by selective oxidation of C-5' of deoxyribose.

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.

Stabilization of neocarzinostatin nonprotein chromophore activity by interaction with apoprotein and with HeLa cells.

The methanol-extracted, nonprotein chromophore of the protein antibiotic neocarzinostatin (NCS), which possesses the full in vitro and in vivo deoxyribonucleic acid (DNA) strand-breaking activities and the ability to inhibit DNA synthesis and growth in HeLa cells of the holoantibiotic, is much more labile to inactivation by heat, 2-mercaptoethanol, long-wavelength UV light, and pH values above 4.8. Inactivation is inversely related to the methanol concentration. The pH activity profile of the isolated chromophore extends to pH values below 7.0. Chromophore inactivation is specifically blocked by the apoprotein of NCS; 100-fold higher concentrations of the apoprotein of another protein antibiotic, auromomycin, gave similar protection, whereas bovine serum albumin is even less effective. The chromophore, and not the apoprotein, is inactivated by heat or light (360 nm) as determined by both activity and isoelectric focusing experiments. In contrast to other chromophoric antibiotic substances (daunorubicin and the extracted chromophore of aurodomomycin), the NCS chromophore interacts irreversibly with HeLa cells at 0 degrees C in serum-free medium so as to inhibit subsequent DNA synthesis at 37 degrees C. Such interaction at 0 degrees C is very rapid, reaching 50% completion in about 15 s, and is not found with native NCS or when apo-NCS is added before the chromophore or when serum is included in the preincubation at 0 degrees C. Washing with apo-NCS or serum-containing (or-free) medium after preincubation of the cells with the chromophore at 0 degrees C fails to reverse the subsequenct inhibition of DNA synthesis.

Gaps in DNA induced by neocarzinostatin bear 3'- and 5'-phosphoryl termini.

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.

Activation and inactivation of neocarzinostatin-induced cleavage of DNA.

The possible role of free radicals in the mechanism of neocarzinostatin (NCS) action was studied. While mercaptene markedly stimulate the ability of NCS to degrade DNA, they also rapidly inactivate the antibiotic in a preincubation and at higher concentration inhibit the degradation reaction. The radiation protector S,2-aminoethylisothiuronium bromide-HBr is the most potent compound tested. Scavengers of diffusible OH radicals, O2- or H2O2 do not result in significant inhibition of the oxygen-dependent cleavage of DNA by NCS; in fact, alcohols and other organic solvents stimulate the reaction several-fold. By contrast, the potent peroxyl free radical scavenger, alpha-tocopherol, blocks the reaction 50% at 50 micron.

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.

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.

Analysis of the two steps in polypeptide chain initiation inhibited by pactamycin.

Earlier work has shown that the inhibition by pactamycin (PM) of polypeptide chain initiation in reticulocyte extracts is associated with (1) a defect in the joining of the 60S subunit to the smaller initiation complex to form an 80S complex ("joining reaction") (Kappen, L. S., Suzuki, H., and Goldberg, I. H. (1973), Proc. Natl. Acad. Sci. U.S.A. 70, 22) and (2) a block after the synthesis of the initial dipeptide (Kappen, L. S., and Goldberg, I. H. (1973), Biochem. Biophys. Res. Commun. 54, 1083). The relative contributions of these two effects to the action of PM and their relationship to one another were evaluated in a system employing sparsomycin that permits both initiation at a certain number of initiation sites and limited oligopeptide formation without termination and release. The degree to which PM blocks the "joining reaction" and leads to the accumulation of 48S initiation complexes that either remain free or are bound to polysomes without the corresponding 60S subunit ("half-mers") was estimated by treatment of polysomes with RNase. Met-tRNAfMet binding factors are required to stabilize the RNase-generated 48S complexes. Under conditions where the initiation factor required for the "joining reaction" functions catalytically, presumably by cycling on and off initiation complexes, PM usually inhibits 80S complex formation 50-70%. Where "joining" is not limiting (presence of at least stoichiometric amounts of joining factor or high Mg2+ concentration) PM leads to the maximal accumulation of the initial dipeptide, Met-Val, in the P-site on the ribosome, indicating a block in a subsequent step in elongation. Binding studies with [3H]PM and the inability of PM to inhibit elongation of preformed Met-Val indicate that PM must interact with the ribosomes at an early stage of initiation. Taken together these data are compatible with the suggestion that PM does not interfere with the ribosomal "joining reaction" per se, but prevents the release and reuse of the joining factor, and in so doing blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome.

Effect of neocarzinostatin-induced strand scission on the template activity of DNA for DNA polymerase I.

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.

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.

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.