New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.     

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.     

Solution NMR structure investigation for releasing mechanism of neocarzinostatin chromophore from the holoprotein

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.     

Selective abstraction of 2H from C-1' of the C residue in AGC.ICT by the radical center at C-2 of activated neocarzinostatin chromophore: structure of the drug/DNA complex responsible for bistranded lesion formation.

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)     

DNA strand scission by neocarzinostatin: molecular recognition process responsible for site-specificity

A series of hexanucleotides possessing A-T, G-C, inosine (I)-C and 2-aminoadenine (ANH2)-T base pairs at 5'-side of the target thymine were prepared and their selectivity for C-5' and C4' oxidation in the NCS-mediated degradation was investigated. Quantitative product analysis indicated that preferential C5' oxidation of deoxyribose moiety of the target T occurs at -5'-AT- and 5'-IT- sites, whereas C5' and C4' oxidation occurs competitively at T of -5'-GT- and -5'-ANH2T- sites. Based on the experimental results, an intercalation model that permits competitive hydrogen abstraction from C5' and C4' of deoxyribose moiety has been proposed.     

Mechanistic studies on the base-catalyzed transformation of neocarzinostatin chromophore: roles of bulged DNA

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.     

Mode of reversible binding of neocarzinostatin chromophore to DNA: evidence for binding via the minor groove

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)     

Induced formation of a DNA bulge structure by a molecular wedge ligand-postactivated neocarzinostatin chromophore

Our previous structure elucidation of the complexes of DNA and postactivated neocarzinostatin chromophore (NCS-chrom) compounds revealed two distinctly different binding modes of this antitumor molecule. A thorough understanding of these results will provide the molecular basis for the binding and DNA chain cleavage properties of NCS-chrom. NCSi-gb is one of the postactivated mimics of NCS-chrom which is formed under thiol-free conditions and is able to bind to DNA. This report describes the structure refinement of the NCSi-gb-bulge-DNA complex [Stassinopoulos, A., Jie, J., Gao, X., and Goldberg, I. H. (1996) Science 272, 1943-1946] and the NMR characterization of the free bulge-DNA and free NCSi-gb. These results reveal that the formation of the complex involves conformational changes in both the DNA and the ligand molecule. Of mechanistic importance for the NCS-chrom-DNA interaction, the two ring systems of the drug are brought closer to each other in the complex. This conformation correlates well with the previously observed marked enhancement of the formation of a DNA bulge cleaving species in the presence of bulge-DNA sequences, due to the promotion of the intramolecular radical quenching of the activated NCS-chrom. Interestingly, the binding of NCSi-gb promotes the formation of a bulge binding pocket; this was not found in the unbound DNA. NCS-chrom is unique among the enediyne antibiotics in its ability to undergo two different mechanisms of activation to form two different DNA binding and cleaving species. The two corresponding DNA complexes are compared. One, the bulge-DNA binder NCSi-gb, involves the major groove, and the second, the duplex binder NCSi-glu which is generated by glutathione-induced activation, involves the minor groove. Since the two NCS-chrom-related ligand molecules contain some common chemical structural elements, such as the carbohydrate ring, the striking differences in their DNA recognition and chain cleavage specificity provide insights into the fundamental principles of DNA recognition and ligand design.     

Binding of the nonprotein chromophore of neocarzinostatin to deoxyribonucleic acid

The methanol-extracted, nonprotein chromophore of neocarzinostatin (NCS), which has DNA-degrading activity comparable to that of the native antibiotic, was found to have a strong affinity for DNA. Binding of chromophore was shown by (1) quenching by DNA of the 440-nm fluorescence and shifting of the emission peak to 420 nm, (2) protection by DNA against spontaneous loss of activity in aqueous solution, and (3) inhibition by DNA of the spontaneous generation of 490-nm fluorescence. Good quantitative correlation was found between these three methods in measuring chromophore binding. There was nearly a 1:1 correspondence between loss of chromophore activity and generation of 490-nm fluorescence, suggesting spontaneous degradation of active chromophore to a highly fluorescent product. Chromophore showed a preference for DNA high in adenine + thymine content in both fluorescence quenching and protection studies. NCS apoprotein, which is known to bind and protect active chromophore, quenched the 440-nm fluorescence, shifted the emission peak to 420 nm, and inhibited the generation of 490-nm fluorescence. Chromophore had a higher affinity for apoprotein than for DNA. Pretreatment of chromophore with 2-mercaptoethanol increased the 440-nm fluorescence seven-fold and eliminated the tendency to generate 490-nm fluorescence. The 440-nm fluorescence of this inactive material was also quenched by DNA and shifted to 420 nm, indicating an affinity for DNA comparable to that of untreated chromophore. However, its affinity for apoprotein was much lower than that of untreated chromophore. Both 2-mercapto-ethanol-treated and untreated chromophore unwound supercoiled pMB9 DNA, suggesting intercalation by both molecules. Since no physical evidence for interaction of native neocarzinostatin with DNA has been found, it is likely that dissociation of the chromophore from the protein and association with DNA are important steps in degradation of DNA by neocarzinostatin.     

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.     

Mode of reversible binding of neocarzinostatin chromophore to DNA: base sequence dependency of binding.

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

Stoichiometric uptake of molecular oxygen and consumption of sulfhydryl groups by neocarzinostatin chromophore bound to DNA

In the presence of DNA, and under conditions which resulted in efficient DNA degradation, the reaction of the neocarzinostatin chromophore with sulfhydryl groups was accompanied by a rapid drop in the oxygen tension of the solution. The total extent of oxygen uptake indicated that, consistently, 1 mol of O2 was consumed/mol of chromophore. The rate of oxygen uptake, however, was strongly dependent on the sulfhydryl concentration, and uptake occurred within a few seconds of the sulfhydryl-induced increase in 420-nm fluorescence of the chromophore. Parallel experiments, in which the sulfhydryl concentration of the solution was monitored, showed that approximately 2 mol of sulfhydryl groups were consumed/mol of chromophore, with kinetics similar to those of O2 uptake. Under anaerobic conditions, only 1 mol of sulfhydryl was consumed, but the sulfhydryl-induced fluorescence increase was not inhibited. These results suggest that (i) a reaction with a single sulfhydryl group converts the chromophore to an activated form, (ii) in the presence of DNA this activated chromophore participates in a subsequent reaction which consumes 1 mol of O2 followed by an additional mole of sulfhydryl, and (iii) each chromophore molecule undergoes only one such reaction cycle. In the absence of sulfhydryl groups, the chromophore slowly degraded, giving a product with intense 490-nm fluorescence. This spontaneous degradation reaction, which does not result in DNA damage, was also accompanied by uptake of nearly 1 mol of O2/mol of chromophore.     

The inhibitory mechanism of in vitro protein phosphorylation by a nonprotein chromophore removed from neocarzinostatin

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.     

Efficacy and site specificity of hydrogen abstraction from DNA 2-deoxyribose by carbonate radicals

The carbonate radical anion CO₃^?? is a potent reactive oxygen species (ROS) produced in vivo through enzymatic one-electron oxidation of bicarbonate or, mostly, via the reaction of CO₂ with peroxynitrite. Due to the vitally essential role of the carbon dioxide/bicarbonate buffer system in regulation of physiological pH, CO₃^?? is arguably one of the most important ROS in biological systems. So far, the studies of reactions of CO₃^?? with DNA have been focused on the pathways initiated by oxidation of guanines in DNA. In this study, low-molecular products of attack of CO₃^?? on the sugar–phosphate backbone in vitro were analyzed by reversed phase HPLC. The selectivity of damage in double-stranded DNA (dsDNA) was found to follow the same pattern C4′ > C1′ > C5′ for both CO₃^?? and the hydroxyl radical, though the relative contribution of the C1′ damage induced by CO₃^?? is substantially higher. In single-stranded DNA (ssDNA) oxidation at C1′ by CO₃^?? prevails over all other sugar damages. An approximately 2000-fold preference for 8-oxoguanine (8oxoG) formation over sugar damage found in our study identifies CO₃^?? primarily as a one-electron oxidant with fairly low reactivity toward the sugar–phosphate backbone.     

Competition between anaerobic covalent linkage of neocarzinostatin chromophore to deoxyribose in DNA and oxygen-dependent strand breakage and base release

Treatment of poly(dA-dT) X poly(dA-dT) with the nonprotein chromophore of neocarzinostatin in the presence of sulfhydryls resulted in both direct and alkali-dependent base release, indicative of DNA sugar oxidation. Covalent chromophore-DNA adducts were also formed. Under anaerobic conditions, base release was strongly inhibited; however, adduct formation was not inhibited and in some cases was markedly enhanced. In the presence of dithiothreitol, anoxia increased adduct formation by a factor of 2, and a particularly stable adduct species was formed, which was recovered from nuclease digests of the treated DNA as a highly fluorescent compound with structure chromophore-d(TpApT). Acid hydrolysis of chromophore-d(TpApT) released free adenine base and both 3'dTMP and 5'dTMP, leaving a compound that contained only chromophore and the deoxyadenosine sugar. These results conclusively confirm that the chromophore forms a covalent adduct with deoxyribose in DNA. Thus, even in the absence of oxygen, activation of the chromophore by sulfhydryls results in the formation of a species capable of reacting with deoxyribose. Several other adduct species were also formed, some of which were nonfluorescent and relatively hydrophilic, but all of which were produced in increased amounts under anoxia. This inverse relation between sugar oxidation and adduct formation suggests that the two lesions share a common precursor. In the presence of other thiols, the effects of anoxia were somewhat different. With glutathione, anoxia markedly enhanced adduct formation, but the total adduct formed was considerably less than with dithiothreitol. With 2-mercaptoethanol, anoxia had no effect on total adduct formation, but the distribution of adduct species was altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.     

Cryospectrokinetic evidence for the mode of reversible binding of neocarzinostatin chromophore to poly(deoxyadenylic-thymidylic acid)

The spectra of neocarzinostatin (NCS) chromophore during its reversible association with poly(dA-dT).poly(dA-dT) [poly(dA-dT)] were recorded (at intervals of 17 ms or more) by a cryospectroscopic method. Examination of the spectral changes of a drug during its interaction with DNA has not been previously reported. Such studies indicate binding of chromophore to poly(dA-dT) is a two-step process in which the spectral properties of the intermediate poly(dA-dT). NCS chromophore species closely resemble those of the final equilibrium species. On the basis of cryokinetic studies (at single wavelengths) carried out at low temperature (2 degrees C), the following proposed mechanism of the DNA-drug (PD) interaction was quantitated: (Formula: see text). In analogy with the other reports on the kinetics of drug-DNA interaction, (PD)I and (PD)II could represent externally bound and intercalated complexes, respectively. However, since the spectra of (PD)I and (PD)II are closely similar, it can also be proposed that (PD)I and (PD)II represent two forms of an intercalated complex. The rate and equilibrium constant for each step were determined by examining the kinetics of the forward and reverse reactions. This was accomplished by determining the polynucleotide concentration dependence of the apparent fast and slow first-order rate constants observed during a double-exponential increase in transmittance (at 330 nm) associated with the binding and the apoprotein-induced dissociation rate constant of the chromophore from poly(dA-dT). The opportunity to use apoprotein, instead of a detergent, to follow the kinetics of the reverse reaction provides a novel approach to these studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

DNA damage by the sulfate radical anion: hydrogen abstraction from the sugar moiety versus one-electron oxidation of guanine

The products of oxidative damage to double-stranded (ds) DNA initiated by photolytically generated sulfate radical anions SO₄^?? were analyzed using reverse-phase (RP) high-performance liquid chromatography (HPLC). Relative efficiencies of two major pathways were compared: production of 8-oxoguanine (8oxoG) and hydrogen abstraction from the DNA 2-deoxyribose moiety (dR) at C1,′ C4,′ and C5′ positions. The formation of 8oxoG was found to account for 87% of all quantified lesions at low illumination doses. The concentration of 8oxoG quickly reaches a steady state at about one 8oxoG per 100 base pairs due to further oxidation of its products. It was found that another guanine oxidation product identified as 2-amino-5-(2′-alkylamino)-4H-imidazol-4-one (X) was released in significant quantities from its tentative precursor 2-amino-5-[(2′-deoxy-β-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) upon treatment with primary amines in neutral solutions. The linear dose dependence of X release points to the formation of dIz directly from guanine and not through oxidation of 8oxoG. The damage to dR was found to account for about 13% of the total damage, with majority of lesions (33%) originating from the C4′ oxidation. The contribution of C1′ oxidation also turned out to be significant (17% of all dR damages) despite of the steric problems associated with the abstraction of the C1′-hydrogen. However, no evidence of base-to-sugar free valence transfer as a possible alternative to direct hydrogen abstraction at C1′ was found.     

A novel o-aminophenol oxidase responsible for formation of the phenoxazinone chromophore of grixazone

Grixazone contains a phenoxazinone chromophore and is a secondary metabolite produced by Streptomyces griseus. In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded. An expression study of GriE and GriF in Escherichia coli showed that GriE activated GriF by transferring copper ions to GriF, as has been observed for a Streptomyces melanogenesis system in which the MelC1 copper chaperon transfers copper ions to MelC2 tyrosinase. In contrast with tyrosinases, GriF showed no monophenolase activity, although it oxidized various o-aminophenols as preferable substrates rather than catechol-type substrates. Deletion of the griEF locus on the chromosome resulted in accumulation of 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) and its acetylated compound, 3-acetylamino-4-hydroxybenzaldehyde. GriF oxidized 3,4-AHBAL to yield an o-quinone imine derivative, which was then non-enzymatically coupled with another molecule of the o-quinone imine to form a phenoxazinone. The coexistence of N-acetylcysteine in the in vitro oxidation of 3,4-AH-BAL by GriF resulted in the formation of grixazone A, suggesting that the -SH group of N-acetylcysteine is conjugated to the o-quinone imine formed from 3,4-AHBAL and that the conjugate is presumably coupled with another molecule of the o-quinone imine. GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.