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1.
Free radical mechanisms in neocarzinostatin-induced DNA damage   总被引:7,自引:0,他引:7  
The molecular mechanisms by which the antitumor protein antibiotic, neocarzinostatin, interacts with DNA and causes DNA sugar damage is discussed. Physical binding of the nonprotein chromophore of neocarzinostatin to DNA, involving an intercalative process and dependent on the microheterogeneity of DNA structure, is followed by thiol activation of the drug to a probable radical species. The latter attacks the deoxyribose, especially at thymidylate residues, by abstracting a hydrogen atom from C-5' to generate a carbon-centered radical on the DNA. This nascent form of DNA damage either reacts with dioxygen to form a peroxyl radical derivative, which eventuates in a strand break with a nucleoside 5'-aldehyde at the 5'-end or reacts with the bound drug to form a novel drug-deoxyribose covalent adduct. Nitroaromatic radiation sensitizers can substitute for dioxygen, but the DNA damage products are different. Similarities between the various biological effects of neocarzinostatin and ionizing radiation are reviewed.  相似文献   

2.
D H Chin  I H Goldberg 《Biochemistry》1986,25(5):1009-1015
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.  相似文献   

3.
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)  相似文献   

4.
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.  相似文献   

5.
Chromatin is the in vivo target site for neocarzinostatin, a DNA strand scission antitumor drug. The effect of neocarzinostatin and its active chromophore component on HeLa cell chromatin is described here. Chromatin consisting of a mixture of mono-, di-, tri- and larger nucleosome fragments is prepared by micrococcal nuclease digestion of HeLa cell nuclei. Drug-induced conversion of chromatin to smaller sized fragments is measured by electrophoresis of the DNA on non-denaturing 4% polyacrylamide gels. Chromatin breakdown measured under these conditions is double-stranded in nature. In the presence of 2 mM dithiothreitol, neocarzinostatin causes degradation of large chromatin fragments and a loss of distinct nucleosome peaks. Detection of chromatin breakdown by neocarzinostatin is dependent upon the concentration of chromatin in the assay. When chromatin is increased from 14 to 70 micrograms/ml, changes in the larger fragments caused by 100 micrograms/ml neocarzinostatin become less obvious are are almost undetectable at 140 micrograms/ml chromatin. No change is observed when chromatin is treated with either neocarzinostatin or its chromophore in the absence of dithiothreitol. For detectable levels of chromatin degradation, 10 micrograms/ml neocarzinostatin is required compared to only 2.5 microgram/ml chromosome (expressed in microgram equivalent neocarzinostatin). Such degradation also occurs more rapidly with chromophore than with neocarzinostatin. Digestion of chromatin with neocarzinostatin continues for at least 30 min at 37 degrees C, while similar degradation caused by chromophore is complete in 1 min. Neocarzinostatin levels which actively degrade isolated chromatin can also effect release of soluble chromatin from intact nuclei. The released chromatin can serve as a substrate for micrococcal nuclease digestion. Such chromatin studies should prove useful in characterizing the mechanism of action of DNA reactive drugs such as neocarzinostatin.  相似文献   

6.
S H Lee  I H Goldberg 《Biochemistry》1989,28(3):1019-1026
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.  相似文献   

7.
The formal C1'-oxidation product, 2-deoxyribonolactone, is formed as a result of DNA damage induced via a variety of agents, including gamma-radiolysis and the enediyne antitumor antibiotics. This alkaline labile lesion may also be an intermediate during DNA damage induced by copper-phenanthroline. Oligo-nucleotides containing this lesion at a defined site were formed via aerobic photolysis of oligonucleotides containing a photolabile ketone, and were characterized by gel electrophoresis and electrospray mass spectrometry (ESI-MS). Treatment of oligo-nucleotides containing the lesion with secondary amines produces strand breaks consisting of 3'-phosphate termini, and products which migrate more slowly in polyacrylamide gels. MALDI-TOF mass spectrometry analysis indicates that the slower moving products are formal adducts of the beta-elimination product resulting from 2-deoxyribonolactone and one molecule of amine. The addition of beta-mercapto-ethanol to the reaction mixture produces thiol adducts as well. The stability of these adducts suggests that they cannot be the labile species characterized by gel electrophoresis in copper-phenanthroline-mediated strand scission. The characterization of these adducts by mass spectrometry also provides, by analogy, affirmation of proposals regarding the reactivity of nucleophiles with the beta-elimination product of abasic sites. Finally, the effects of this lesion and the various adducts on DNA repair enzymes are unknown, but their facile generation from oligonucleotides containing a photolabile ketone suggests that such issues could be addressed.  相似文献   

8.
Bleomycin (BLM) binding and chemistry are apparently sensitive to differences in nucleic acid conformation and could conceivably be developed as a probe for sequence-dependent elements of conformation. We report on the development of a new methodology to synthesize heterogeneous DNA-RNA hybrids of defined sequence and present the results of our comparative studies on the cleavage of DNA and DNA-RNA hybrids by four drugs: BLM, neocarzinostatin and esperamicins A1 and C. In the case of BLM with duplex DNA, purine-pyrimidine steps such as GT and GC, are consistently hit, as previously observed. However, in heterogeneous sequence hybrids, not all GC sites are recognized by the drug, although all GT sites are. Suppressed GC sites are consistently flanked by pyrimidines on both the 3' and 5' sides, suggesting that the BLM binding site in hybrids spans at least four bases. Kinetic isotope studies with specifically deuterated substrates (kH/kD = 1.2-4.0) and the effect of oxygen on the product profile are presented in support of a mechanism consistent with 4'-hydrogen abstraction in hybrids. The powerful double-labeled probe technique was extended to study the mechanism of action of other DNA degrading drugs on DNA-RNA hybrids. For neocarzinostatin, the sequence specificity lies in the AT-rich region for hybrids and is similar to that of DNA, however, the overall cleavage pattern for the hybrid is significantly different from that for the same sequence of DNA. In the hybrid, a stretch of AT residues is essential and the A sites are damaged to a greater extent than they are in DNA. However, no kinetic isotope effects are observed and, based on the product profile, the mechanism of degradation of the DNA strand of hybrids seems to be limited to abstraction of the 5'-hydrogen. For esperamicin A1, damage on the DNA strand of hybrids occurs exclusively via 5'-hydrogen abstraction in a non-rate determining step and primarily at A and T sites. Esperamicin C behaves similarly, exhibiting no isotope effects at 1', 4' and 5' positions. Overall, the differences observed in site-specific cleavage between the two substrates is proposed to be a result of conformational differences between the DNA strand of duplex DNA and DNA-RNA hybrids.  相似文献   

9.
D Dasgupta  I H Goldberg 《Biochemistry》1985,24(24):6913-6920
Two general approaches have been taken to understand the mechanism of the reversible binding of the nonprotein chromophore of neocarzinostatin to DNA: (1) measurement of the relative affinity of the chromophore for various DNAs that have one or both grooves blocked by bulky groups and (2) studies on the influence of adenine-thymine residue-specific, minor groove binding agents such as the antibiotics netropsin and distamycin on the chromophore-DNA interaction. Experiments using synthetic DNAs containing halogen group (Br, I) substituents in the major groove or natural DNAs with glucosyl moieties projecting into the major groove show that obstruction of the major groove does not decrease the binding stoichiometry or the binding constant for the DNA-chromophore interaction. Chemical methylation of bases in both grooves of calf thymus DNA, resulting in 13% methylation of N-7 of guanine in the major groove and 7% methylation of N-3 of adenine in the minor groove, decreases the binding affinity and increases the size of the binding site for neocarzinostatin chromophore. Similar results were obtained whether binding parameters were determined directly by spectroscopic measurements or indirectly by measuring the ability of the DNA to protect the chromophore against degradation. On the other hand, netropsin and distamycin compete with neocarzinostatin chromophore for binding to the minor groove of DNA, as shown by their decrease in the ability of poly(dA-dT) to protect the chromophore against degradation and their reduction in chromophore-induced DNA damage as measured by thymine release.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

10.
Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.  相似文献   

11.
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.  相似文献   

12.
Strand scission of DNA by the chromophore of neocarzinostatin converts the 5'-hydroxyl of deoxyribose to a 5'-aldehyde. The origin of the aldehydic oxygen has now been elucidated by mass spectrometry. DNA-associated thymidine 5'-aldehyde produced by treatment of DNA with neocarzinostatin chromophore in 2H218O/16O2 or in 2H216O/18O2 was reduced, liberated by nuclease treatment, permethylated, and analyzed by gas chromatography-mass spectrometry. The data clearly show that molecular oxygen is the only source of the 5'-aldehydic oxygen. The addition of molecular oxygen at C-5', possibly via a reactive form of neocarzinostatin chromophore, must be involved; a carbonium ion intermediate at C-5' is ruled out.  相似文献   

13.
L F Povirk  I H Goldberg 《Biochemistry》1985,24(15):4035-4040
A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs.  相似文献   

14.
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.  相似文献   

15.
Neocarzinostatin is a potent antitumor antibiotic and is a prodrug, which induces genome damage after activation by a thiol. The prodrug is stored as a protein-bound chromophore that contains an enediyne nucleus. A thiolate attack on the chromophore cyclizes the nucleus and produces radicals that abstract hydrogen from DNA. Because thiol is the only cofactor in the vital activation process, the structure of the thiol plays an important role in the activity of the drug. Here we systematically examine the effect of the electronic structure of some thiols on the efficiency of the drug, and compare particularly aromatic with aliphatic thiols. The values of drug-induced base release from DNA are remarkably different between thiophenol (3.6%) and benzyl mercaptan (12.5%), the activity of which is comparable with those of aliphatic thiols. Cleavage results determined by DNA electrophoresis are consistent with the results of base release; they show that the total number of DNA lesions is more than 3-fold lower for thiophenol than for aliphatic thiols or benzyl mercaptan. We conclude that among aromatic thiols, only those that have delocalized thiol sulfur electrons can substantially reduce the DNA cleavage activity. This result suggests that the effect of an aromatic ring arises from an inductive effect imposed on the thiol sulfur electron through pi-resonance rather than through effects such as aromatic stacking, steric hindrance, or hydrophobic interaction. Replacing thiophenol with substituted derivatives with electron-releasing or -withdrawing groups changes the drug activity and supports the important role of the electronic structure of the thiol sulfur in determining the drug activity.  相似文献   

16.
Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.  相似文献   

17.
The leafy parts of thyme and its essential oil have been used in foods for its flavour, aroma and preservation for many years. In the present study the genotoxic potential of major compounds of thyme oil, i.e. thymol, carvacrol, and gamma-terpinene and of the methanolic extracts of thyme, were investigated in human lymphocytes by single-cell gel electrophoresis. Also, the effects of these substances on the induction of DNA damage by 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and mitomycin C (MMC) were evaluated. No increase in DNA strand breakage was observed at thymol and gamma-terpinene concentrations below 0.1 mM, but at the higher concentration of 0.2 mM significant increases in DNA damage were seen. Thymol and gamma-terpinene significantly reduced the DNA strand breakage induced by IQ and MMC at the lower concentrations studied. Carvacrol, which is an isomer of thymol, seemed to protect lymphocytes from the genotoxic effects of IQ and MMC at non-toxic concentrations below 0.05 mM, but at the higher concentration of 0.1 mM carvacrol itself induced DNA damage. Also the constituents of the n-hexane and ethyl acetate fractions prepared from the concentrated aqueous methanolic extracts of Thymus spicata protected lymphocytes against IQ- and MMC-induced DNA damage in a concentration-dependent manner.  相似文献   

18.
Xi Z  Mao QK  Goldberg IH 《Biochemistry》1999,38(14):4342-4354
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.  相似文献   

19.
The inhibitory effect of a nonprotein chromophore removed from neocarzinostatin on protein phosphorylation by nuclear protein kinase in vitro has been studied. Low levels of the chromophore greatly inhibited protein phosphorylation in vitro. This inhibition, however, was not selectively dependent on the indicated kinases and their different phosphate acceptors (histones and non-histone protein). In contrast, the protein component (apoprotein) of neocarzinostatin did not affect the phosphorylation even at a concentration of 400-times higher than that of the chromophore. Moreover, apoprotein suppressed the chromophore-induced inhibition of protein phosphorylation in vitro in proportion to the apoprotein concentrations. Kinetic and analytical experiments suggest that the chromophore-induced inhibition of protein phosphorylation seems to be due to the binding of the chromophore to the kinases. In addition, we found that ultraviolet irradiation as well as methanol extraction can release the chromophore from neocarzinostatin, but it exhibits no inhibitory activity of DNA synthesis in growing cells. The fact that the chromophore-induced inhibition of protein phosphorylation in vitro was not sensitive to ultraviolet irradiation, which rapidly inactivated the ability of the chromophore to induce DNA degradation in vitro, suggests that there are different actions involved in the two inhibitions induced by the chromophore which is removed from neocarzinostatin.  相似文献   

20.
L S Kappen  I H Goldberg 《Biochemistry》1983,22(21):4872-4878
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.  相似文献   

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