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 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').
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)
The methanol-extractable, nonprotein chromophore of the antitumor, protein antibiotic neocarzinostatin (NCS) has at least the full activity of the parent compound in inhibiting DNA synthesis and growth of HeLa cells and in causing DNA strand breaks in vivo and in vitro. In vitro DNA strand scission by the chromophore is markedly stimulated by 2-mercaptoethanol and is inhibited by guanidine hydrochloride and a-tocopherol. By high-pressure liquid chromatography, this activity has been localized to fractions eluting at >90% methanol and having fluorescence emission at 420 nm (excitation at 340 nm). The apo-protein of NCS is inactive by itself but complexes with the chromophore so as to regulate its availability during the in vitro reaction. In DNA strand scission the chromophore acts rapidly at both 0 and 370C, whereas native and reconstituted NCS are inactive at 00C and slowly active at 370C.Complex formation with apo-NCS stabilizes the chromophore.Reconstitution of NCS (pI 3.3) from chromophore and apo-protein (pI 3.2) was shown by both activity studies and isoelectric focusing on polyacrylamide gels. "Pre-NCS," the biosynthetic precursor of NCS, is identical to apo-NCS in amino acid composition, spectral properties, isoelectric focusing on polyacrylamide gels, and ability to complex with isolated chromophore to form material with all the properties of native NCS. Neocarzinostatin (NCS), an antitumor antibiotic, is a singlechain, acidic protein of molecular weight 10,700 with two disulfide bonds; its amino acid sequence is known (1). Considerable evidence has been presented to indicate that cellular DNA is the major target in the action of NCS and that the drug causes DNA strand breakage both in vivo and in vitro (reviewed in ref.2). NCS introduces single-strand breaks almost exclusively at thymidylate and adenylate residues in DNA in vitro (3-5) in a reaction greatly stimulated by a sulfhydryl compound (3, 6-9) and dependent on oxygen (9-11). Evidence for the existence of an active, labile form of NCS that causes single-strand breaks in linear and supercoiled DNA has been presented (9, 12). With the recent demonstration that NCS contains nonprotein, chromophoric material that has UV-visible absorption above 300 nm and fluorescence emission at 420 nm and 490 nm and that can be separated from the protein (13), the question arises as to its possible role in NCS action. Indirect evidence for such a role comes from our finding that NCS protein from which the chromophore(s) has been removed or destroyed by various procedures has lost virtually all of its biological activity (12,14).In this paper we offer direct evidence that the isolated (15) and has the same amino acid composition as NCS, although deamidation of the asparagine in position 83 has been reported (16). The UV-visible absorption and fluorescence spectra of the "pre-NCS" were found to be identical to those reported for apo-NCS (13,14). Comparison of the biological properties of NCS and macromomycin has been reported (12, 17). The assays for NCS-i...
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.
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