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.     

Probing DNA bulges with designed helical spirocyclic molecules

Because bulged structures (unpaired bases) in nucleic acids are of general biological significance, it has been of interest to design small molecules as specific probes of bulge function. On the basis of our earlier work with the specific DNA bulge-binding metabolite obtained from the enediyne antitumor antibiotic neocarzinostatin chromophore (NCS-chrom), we have prepared three small helical spirocyclic molecules that most closely mimic the natural product. These wedge-shaped molecules resemble the natural product in having the sugar residue attached to the same five-membered ring system. In one instance, the sugar is aminoglucose in beta-glycosidic linkage, and in the other, two enantiomers have the natural sugar N-methylfucosamine in alpha-glycosidic linkage. All three analogues were found to interfere with bulge-specific cleavage by NCS-chrom and the ability of bulged DNA to serve as a template for DNA polymerase 1 in accordance with their binding affinities for DNA containing a two-base bulge. Comparable results were obtained with the analogues for the less efficiently cleaved three-base bulge DNA structures. In each situation, the enantiomers possessing the natural sugar in alpha-glycosidic linkage are the most potent inhibitors of the cleavage reaction. In the DNA polymerase reactions, again, the closest natural product mimics were the most effective in selectively impeding nucleotide extension at the bulge site, presumably by complex formation. These results demonstrate the potential usefulness of bulge-binding compounds in modifying DNA structure and function and support efforts to design and prepare reactive species of these molecules that can covalently modify bulged DNA.     

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.     

Stereochemical control of small molecule binding to bulged DNA: comparison of structures of spirocyclic enantiomer-bulged DNA complexes

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and ent-DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. Ent-DDI is a left-handed wedge-shaped spirocyclic molecule whose aglycone portion is an enantiomer of DDI, which mimics the spirocyclic geometry of the natural product, NCSi-gb, formed by base-catalyzed activation of the enediyne antibiotic neocarzinostatin. The benzindanone moiety of ent-DDI intercalates between the A6.T21 and the T9.A20 base pairs, overlapping with portions of the purine bases; the dihydronaphthalenone moiety is positioned in the minor groove along the G7-T8-T9 bulge sequence; and the aminoglycoside is in the middle of the minor groove, approaching A20 of the nonbulged strand. This alignment of ent-DDI along the DNA helical duplex is in the reverse direction to that of DDI. The aminoglycoside moiety of ent-DDI is positioned in the 3' direction from the bulge region, whereas that of the DDI is positioned in the 5' direction from the same site. This reverse binding orientation within the bulge site is the natural consequence of the opposite handedness imposed by the spirocyclic ring junction and permits the aromatic ring systems of the two spirocyclic enantiomers access to the bulge region. NMR and CD data indicate that the DNA in the DDI-bulged DNA complex undergoes a larger conformational change upon complex formation in comparison to the ent-DDI-bulged DNA, explaining the different binding affinities of the two drugs to the bulged DNA. In addition, there are different placements of the bulge bases in the helical duplex in the two complexes. One bulge base (G7) stacks inside the helix, and the other one (T8) is extrahelical in the DDI-bulged DNA complex, whereas both bulge bases in the ent-DDI-bulged DNA complex prefer extrahelical positions for drug binding. Elucidation of the detailed binding characteristics of the synthetic spirocyclic enantiomers provides a rational basis for the design of stereochemically controlled drugs for bulge binding sites.     

Solution structure of a wedge-shaped synthetic molecule at a two-base bulge site in DNA

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. DDI, which has been found to modulate DNA strand slippage synthesis by DNA polymerase I [Kappen, L. S., Xi, Z., Jones, G. B., and Goldberg, I. H. (2003) Biochemistry 42, 2166-2173], is a wedge-shaped spirocyclic molecule whose aglycone structure closely resembles that of the natural product, NCSi-gb, which strongly binds to an oligonucleotide containing a two-base bulge. Changes in chemical shifts of the DNA upon complex formation and intermolecular NOEs between DDI and the bulged DNA duplex indicate that agent specifically binds to the bulge site of DNA. The benzindanone moiety of DDI intercalates via the minor groove into the G7-T8-T9.A20 pocket, which consists of a helical base pair and two unpaired bulge bases, stacking with the G7 and A20 bases. On the other hand, the dihydronaphthalenone and aminoglycoside moieties are positioned in the minor groove. The aminoglycoside, which is attached to spirocyclic ring, aligns along the A20T21G22 sequence of the nonbulged strand, while the dihydronaphthalenone, which is restrained by the spirocyclic structure, is positioned near the G7-T8-T9 bulge site. The aminoglycoside is closely aligned with the dihydronaphthalenone, preventing its intercalation into the bulge site. In the complex, the unpaired purine (G7) is intrahelical and stacks with the intercalating moiety of DDI, whereas the unpaired pyrimidine (T8) is extrahelical. The structure of the complex formed by binding of the synthetic agent to the two-base bulged DNA reveals a binding mode that differs in important details from that of the natural product, explaining the different binding specificity for the bulge sites of DNA. The structure of the DDI-bulged DNA complex provides insight into the structure-binding affinity relationship, providing a rational basis for the design of specific, high-affinity probes of the role of bulged nucleic acid structures in various biological processes.     

Solution structure of a designed spirocyclic helical ligand binding at a two-base bulge site in DNA

The solution structure of the complex formed between an oligodeoxynucleotide containing a two-base bulge (5'-CCATCGTCTACCTTTGGTAGGATGG) and SCA-alpha2, a designed spirocyclic helical molecule, has been elucidated. SCA-alpha2, a close mimic of the metabolite, NCSi-gb, of the DNA bulge-specific enediyne antibiotic neocarzinostatin, differs in possessing a more stable spirocyclic ring system and in lacking certain bulky groupings that compromise bulged DNA binding. This study provides a detailed comparison of the binding modes of the two complexes and provides new insights into the importance of shape and space, as opposed to simple nucleotide sequence, in complex formation at the bulge site. The two rigidly held aromatic rings of SCA-alpha2 form a right-handed helical molecular wedge that specifically penetrates the bulge-binding pocket and immobilizes the two bulge residues (GT), which point toward the minor groove, rather than the major groove as in the NCSi-gb.bulged DNA complex. The ligand aromatic ring systems stack on the DNA bulge-flanking base pairs that define the long sides of the triangular prism binding pocket. Like NCSi-gb, SCA-alpha2 possesses the natural N-methylfuranose moiety, alpha-linked to the benzindanol (BI) moiety. The amino sugar anchors in the major groove of the DNA and points toward the 3'-bulge-flanking base pair. Lacking the bulky cyclocarbonate of NCSi-gb, the SCA-alpha2.bulged DNA complex has a much less twisted and buckled 3'-bulge-flanking base pair (dG20.dC8), and the G20 residue stacks directly above the BI ring platform. Also, the absence of the methyl group and the free rotation of the methoxy group on the dihydronaphthanone (NA) moiety of SCA-alpha2 allow better stacking geometry of the NA ring above the 5'-bulge-flanking dG21.dC5 base pair. These and other considerations help to explain why NCSi-gb binds very poorly to bulged RNA and are consistent with the recent observation of good binding with SCA-alpha2. Thus, although the two complexes resemble each other closely, they differ in important local environmental details. SCA-alpha2 has a better hand-in-glove fit at the bulge site, making it an ideal platform for the placement of moieties that can react covalently with the DNA and for generating congeners specific for bulges in RNA.     

Specific binding of 2-amino-1,8-naphthyridine into a single guanine bulge as evidenced by photooxidation of GG doublet

Photoirradiation of 2-amino-1,8-naphthyridines in the presence of duplex DNA containing the GG doublet opposite a single bulge was examined. After hot piperidine treatment, DNA cleavage was observed preferentially at the GG opposite a single bulge. The cleavage efficiency was highly dependent on the nature of bulged base. The G cleavage at the GG opposite a single G bulge was exceptionally weak, suggesting an intercalative binding of 2-amino-1,8-naphthyridine chromophore into the GG step.     

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.     

Designed DNA probes from the neocarzinostatin family: Impact of glycosyl linkage stereochemistry on bulge base binding

Bulged sites in DNA and RNA have become targets for rational drug design due to their suspected involvement in a number of key biomolecular processes. A lead compound, derived from the enediyne natural product NCS-chrom has been used to inform chemical synthesis of a family of designed probes of DNA bulges, one of which shows 80 nM affinity for a two base bulged target. Key contributors to binding of these spirocyclic compounds have been studied in order to correlate affinity and specificity with structural features. Herein, we demonstrate that the glycosyl linkage stereochemistry of the pendant aminofucosyl group plays a pivotal role in binding, and coupled with insight obtained with various bulged targets, will allow rational design of second generation ligands.     

Recognition of bulged DNA by a neocarzinostatin product via an induced fit mechanism

The binding of the wedge-shaped isostructural analogue of the biradical species of the chromphore of antitumor antibiotic neocarzinostatin to sequence-specific bulged DNAs results in alterations in ellipticity of the DNAs. Circular dichroism (CD) spectroscopic results suggest that the drug specifically recognizes bulges of DNA via a combination of conformational selection and induced fit, not by binding to a preorganized site. Analysis of circular dichroism spectra indicates that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the bulge cavity. The effective recognition of the bulge site on duplex DNA appears to depend to a significant extent on the bent groove space being flexible enough to be able to adopt the geometrically optimal conformation compatible with the wedge-shaped drug molecule, rather than involving 'lock and key' recognition. The spectroscopic results indicate a change of DNA conformation, consistent with an allosteric binding model. Spectroscopic studies with various bulged DNAs also reveal that the binding strength directly correlates with the stability of the bulge structures.     

DNA damage by thiol-activated neocarzinostatin chromophore at bulged sites

Bulge structures in nucleic acids are of general biological significance and are potential targets for therapeutic drugs. It has been shown in a previous study that thiol-activated neocarzinostatin chromophore is able to cleave duplex DNA selectively at a position opposite a single unpaired cytosine or thymine base on the 3' side. In this work, we studied in greater detail the nature of this type of cleavage and the basis for the selectivity of the bulge site cleavage over the usual strand cleavage at a T site in the duplex region by using duplexes containing an internal control and a bulge, which is composed of different types and number of bases. Experimental results indicated that the bulge-induced cleavage is initiated by 5' hydrogen abstraction and is greatly affected by the base composition of the bulge. A single-base bulge, especially when containing a purine, yields higher efficiency and greater selectivity for the bulge-induced cleavage. In particular, a single adenine base gives rise to the highest cleavage yield and provides over 20 times greater selectivity for cleavage at the bulge site compared with the internal control site in duplexes. The binding dissociation constants of postactivated drug for a stem-loop structure containing a one- or two-base bulge in the stem, measured by fluorescence quenching, show that the binding is about 3-4 times stronger for bulge-containing duplexes than for perfect hairpin duplexes. For RNA.DNA hybrid duplexes, where the DNA is the target strand and the RNA is the bulge-containing strand, bulge-induced cleavage was observed, although at low yield. On the other hand, when RNA is the nonbulge strand, no bulge-induced cleavage was found. When the reaction is performed in the absence of oxygen, the major product is a covalent adduct, and it is at the same location as the cleavage site under aerobic conditions.     

X-ray crystallographic observation of "in-line" and "adjacent" conformations in a bulged self-cleaving RNA/DNA hybrid

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Hüsken et al., Biochemistry, 1996, 35:16591-16600). Transesterification proceeds via an in-line mechanism, with the 2'-OH of the bulged nucleotide attacking the 3'-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2'-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2'-oxygen of the bulged nucleotide and the P-O5' bond (including adjacent and near in-line) and give a detailed picture of the conformational changes necessary to line up the 2'-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308-1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.     

Development of new simple molecular probes of DNA bulged structures

NCSi-gb is a neocarzinostatin chromophore (NCS-chrom) metabolite which binds strongly to certain two-base DNA bulges. Compared with previously reported NCSi-gb analogues, a new analogue with a different aminoglycoside position was synthesized, and it showed strong fluorescence and improved binding and sequence selectivity to DNA bulges. The N-dimethylated form of this analogue had a similar binding pattern, and it competitively inhibited bulge-specific cleavage by NCS-chrom.     

Comparative reactivity of mismatched and unpaired bases in relation to their type and surroundings. Chemical cleavage of DNA mismatches in mutation detection analysis

Systematic study of chemical reactivity of non-Watson–Crick base pairs depending on their type and microenvironment was performed on a model system that represents two sets of synthetic DNA duplexes with all types of mismatched and unmatched bases flanked by T·A or G·C pairs. Using comparative cleavage pattern analysis, we identified the main and additional target bases and performed quantitative study of the time course and efficacy of DNA modification caused by potassium permanganate or hydroxylamine. Potassium permanganate in combination with tetraethylammonium chloride was shown to induce DNA cleavage at all mismatched or bulged T residues, as well as at thymines of neighboring canonical pairs. Other mispaired (bulged) bases and thymine residues located on the second position from the mismatch site were not the targets for KMnO₄ attack. In contrast, hydroxylamine cleaved only heteroduplexes containing mismatched or unmatched C residues, and did not modify adjacent cytosines. However when G·C pairs flank bulged C residue, neighboring cytosines are also attacked by hydroxylamine due to defect migration. Chemical reactivity of target bases was shown to correlate strongly with the local disturbance of DNA double helix at mismatch or bulge site. With our model system, we were able to prove the absence of false-negative and false-positive results. Portion of heteroduplex reliably revealed in a mixture with corresponding homoduplex consists of 5% for bulge bases and “open” non-canonical pairs, and 10% for wobble base pairs giving minimal violations in DNA structure. This study provides a complete understanding of the principles of mutation detection methodology based on chemical cleavage of mismatches and clarifies the advantages and limitations of this approach in various biological and conformational studies of DNA.     

The photochemistry of 8-bromo-2'-deoxyadenosine. A direct entry to cyclopurine lesions.

The UV photolysis of 8-bromo-2'-deoxyadenosine has been investigated in different solvents and in the presence of additives like halide anions. Photolytic cleavage of the C-Br bond leads to formation of the C8 radical. In methanol, subsequent hydrogen abstraction from the solvent is the main radical reaction; however, in water or acetonitrile intramolecular hydrogen abstraction from the sugar moiety, to give the C5' radical, is the major path. This C5' radical undergoes a cyclization reaction on the adenine and gives the aminyl radical. A rate constant of 1.8 x 10(5) s(-1) has been measured by laser flash photolysis in CH(3)CN for this unimolecular process. Product studies from steady-state photolysis in acetonitrile have shown the conversion of 8-bromo-2'-deoxyadenosine to 5',8-cyclo-2'-deoxyadenosine in 65% yield and in a diastereoisomeric ratio (5' R):(5' S)= 1.7. Evidence supporting that the equilibrium Br*+ Br(-)[right left harpoons] Br(2)*(-) plays an important role in this synthetically useful radical cascade is obtained by regulating the relative concentrations of the two reactive oxidizing species.     

Dioxygenases without requirement for cofactors and their chemical model reaction: compulsory order ternary complex mechanism of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase involving general base catalysis by histidine 251 and single-electron oxidation of the substrate dianion

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the alpha/beta hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k(cat)/K(m,A) and k(cat)/K(m,O)()2, were 1.4 x 10(8) and 3.0 x 10(5) M(-1) s(-1) with I and 1.2 x 10(5) and 0.41 x 10(5) M(-1) s(-1) with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the alpha/beta hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D(2)O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.     

Molecular probes of DNA bulges: functional assay and spectroscopic analysis

Bulged structures in DNA and RNA have been linked to biomolecular processes involved in numerous diseases, thus probes with affinity for these nucleic acid targets would be of considerable utility to chemical biologists. Herein, we report guided chemical synthesis of small molecules capable of binding to DNA bulges by virtue of their unique (spirocyclic) geometry. The agents, modeled on a natural product congener, show pronounced selectivity for specific bulged motifs and are able to enhance slipped DNA synthesis, a hallmark functional assay of bulge binding. Significantly, bulge-agent complexes demonstrate characteristic fluorescent signatures depending on bulge and flanking sequence in the oligo. It is anticipated that these signature patterns can be harnessed as molecular probes of bulged hotspots in DNA and RNA.     

Selective strand scission by intercalating drugs at DNA bulges

A bulge is an extra, unpaired nucleotide on one strand of a DNA double helix. This paper describes bulge-specific strand scission by the DNA intercalating/cleaving drugs neocarzinostatin chromophore (NCS-C), bleomycin (BLM), and methidiumpropyl-EDTA (MPE). For this study we have constructed a series of 5'-32P end labeled oligonucleotide duplexes that are identical except for the location of a bulge. In each successive duplex of the series, a bulge has been shifted stepwise up (from 5' to 3') one strand of the duplex. Similarly, in each successive duplex of the series, sites of bulge-specific scission and protection were observed to shift in a stepwise manner. The results show that throughout the series of bulged duplexes NCS-C causes specific scission at a site near a bulge, BLM causes specific scission at a site near a bulge, and MPE-Fe(II) causes specific scission centered around the bulge. In some sequences, NCS-C and BLM each cause bulge-specific scission at second sites. Further, bulged DNA shows sites of protection from NCS-C and BLM scission. The results are consistent with a model of bulged DNA with (1) a high-stability intercalation site at the bulge, (2) in some sequences, a second high-stability intercalation site adjacent to the first site, and (3) two sites of relatively unstable intercalation that flank the two stable intercalation sites. On the basis of our results, we propose a new model of the BLM/DNA complex with the site of intercalation on the 3' side (not in the center) of the dinucleotide that determines BLM binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sites in the diyne-ene bicyclic core of neocarzinostatin chromophore responsible for hydrogen abstraction from DNA

The antitumor antibiotic neocarzinostatin exhibits its main drug action by abstracting hydrogen from DNA deoxyribose with consequent strand breakage or related lesions. All biological activities of the drug derive solely from a nonprotein chromophoric substance (NCS-chrom) consisting of a novel epoxy-bicyclo-diyne-ene system. Thiol or sodium borohydride activates NCS-chrom into a labile, reactive species that induces DNA damage but causes inactivation of the drug in the absence of the target DNA. Mass spectrometric studies indicate that the isolated thiol-activated NCS-chrom product in the presence of DNA has the same molecular weight as the thiol-inactivated NCS-chrom product in the absence of DNA. No deuterium is incorporated into the chromophore from the deuterium-labeled sulfhydryl group. Since three deuterium atoms can be incorporated into the drug by treatment with sodium borodeuteride without DNA, adding an unlabeled DNA under parallel conditions permitted the ready identification of the activated NCS-chrom product that abstracted hydrogen from the DNA. Not only does the activated NCS-chrom product have the same structure as the inactivated drug without DNA, but two of the incorporated deuterium atoms have been substituted by hydrogen. With the aid of NMR spectrometry, the two replaced hydrogen atoms are found to be incorporated into the C-2 and C-6 positions of the bicyclo-diyne-ene ring of NCS-chrom and are derived neither from borodeuteride nor from the hydroxyl functions of the solvents. In accord with current proposals, the two hydrogens incorporated into the drug may come from closely opposed sites on the complementary strands of the DNA at which the drug is bound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positional effect of single bulge nucleotide on PNA(peptide nucleic acid)/DNA hybrid stability.

We report positional effect of bulge nucleotide on PNA/DNA hybrid stability. CD spectra showed that PNA/DNA hybrids required at least seven base pairings at a stem region to form a bulged structure. On the other hand, DNA/DNA could form bulged structure when there are only four base pairings adjacent to the bulge nucleotide. We discuss why PNA requests such a many base pairings to form bulged structure from a nearest neighbor standpoint.