In vitro expansion of DNA triplet repeats with bulge binders and different DNA polymerases

The expansion of DNA repeat sequences is associated with many genetic diseases in humans. Simple bulge DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. To probe the possible role of bulged structures in DNA slippage, we designed and synthesized a pair of simple chiral spirocyclic compounds [Xi Z, Ouyang D & Mu HT (2006) Bioorg Med Chem Lett 16, 1180-1184], DDI-1A and DDI-1B, which mimic the molecular architecture of the enediyne antitumor antibiotic neocarzinostatin chromophore. Both compounds strongly stimulated slippage in various DNA repeats in vitro. Enhanced slippage synthesis was found to be synchronous for primer and template. CD spectra and UV thermal stability studies supported the idea that DDI-1A and DDI-1B exhibited selective binding to the DNA bulge and induced a significant conformational change in bulge DNA. The proposed mechanism for the observed in vitro expansion of long DNA is discussed.     

Stimulation on DNA triplet repeat strand slippage synthesis by the designed spirocycles

The designed simpler chiral spirocyclic helical compounds that mimic the molecular architecture of the DNA bulge binder NCSi-gb have been prepared. It has been found that the synthesized spirocyclic compounds have strong stimulation effect on DNA slippage synthesis. Their stimulation activities on DNA strand slippage suggest that they may bind to or induce the formation of a non Watson-Crick structure during in vitro replication of DNA triplet repeats.     

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 dAATAA and dAAUAA DNA bulges

The NMR structure analysis is described for two DNA molecules of identical stem sequences with a five base loop containing a pyrimidine, thymin or uracil, in between purines. These five unpaired nucleotides are bulged out and are known to induce a kink in the duplex structure. The dAATAA bulge DNA is kinked between the third and the fourth nucleotide. This contrasts with the previously studied dAAAAA bulge DNA where we found a kink between the fourth and fifth nucleotide. The total kinking angle is ~104° for the dAATAA bulge. The findings were supported by electrophoretic data and fluorescence resonance energy transfer measurements of a similar DNA molecule end-labeled by suitable fluorescent dyes. For the dAAUAA bulge the NMR data result in a similar structure as reported for the dAATAA bulge with a kinking angle of ~87°. The results are discussed in comparison with a rAAUAA RNA bulge found in a group I intron. Generally, the sequence-dependent structure of bulges is important to understand the role of DNA bulges in protein recognition.     

Structural Insights Into DNA Repair by RNase T—An Exonuclease Processing 3′ End of Structured DNA in Repair Pathways

DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3′ end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3′ end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3′ overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V–dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3′-end processing.     

Bulge oligonucleotide as an inhibitory agent of bacterial topoisomerase I

Bacterial topoisomerase I (Btopo I) was defined as potential target for discovery of new antibacterial compounds. Various oligonucleotides containing bulge structure were designed and synthesised as inhibitors to Btopo I in this investigation. The results of this study demonstrated that the designed oligonucleotides display high inhibitory efficiency on the activity of Btopo I and the inhibitory effect could be modulated by the amount of bulge DNA bases. The most efficient one among them showed an IC₅₀ value of 63.1?nM in its inhibition on the activity of Btopo I. In addition, our studies confirmed that the designed oligonucleotide would induce irreversible damages to Btopo I and without any effects occur to eukaryotic topoisomerase I. It is our hope that the results provided in these studies could provide a novel way to inhibit Btopo I.     

Role of the CCA bulge of prohead RNA of bacteriophage ø29 in DNA packaging

The oligomeric ring of prohead RNA (pRNA) is an essential component of the ATP-driven DNA packaging motor of bacteriophage ?29. The A-helix of pRNA binds the DNA translocating ATPase gp16 (gene product 16) and the CCA bulge in this helix is essential for DNA packaging in vitro. Mutation of the bulge by base substitution or deletion showed that the size of the bulge, rather than its sequence, is primary in DNA packaging activity. Proheads reconstituted with CCA bulge mutant pRNAs bound the packaging ATPase gp16 and the packaging substrate DNA-gp3, although DNA translocation was not detected with several mutants. Prohead/bulge-mutant pRNA complexes with low packaging activity had a higher rate of ATP hydrolysis per base pair of DNA packaged than proheads with wild-type pRNA. Cryoelectron microscopy three-dimensional reconstruction of proheads reconstituted with a CCA deletion pRNA showed that the protruding pRNA spokes of the motor occupy a different position relative to the head when compared to particles with wild-type pRNA. Therefore, the CCA bulge seems to dictate the orientation of the pRNA spokes. The conformational changes observed for this mutant pRNA may affect gp16 conformation and/or subsequent ATPase-DNA interaction and, consequently, explain the decreased packaging activity observed for CCA mutants.     

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.     

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)     

Structure of the RNA claw of the DNA packaging motor of bacteriophage ?29

Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage ϕ29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33–35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.     

Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP

DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.     

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.     

Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.     

Stabilization factors affecting duplex formation of peptide nucleic acid with DNA.

Peptide nucleic acid (PNA) is an oligonucleotide analogue in which the sugar-phosphate backbone is replaced by an N-(2-aminoethyl)glycine unit to which the nucleobases are attached. We investigated the thermodynamic behavior of PNA/DNA hybrid duplexes with identical nearest neighbors but with different sequences and chain lengths (5, 6, 7, 8, 10, 12, and 16 mers) to reveal whether the nearest-neighbor model is valid for the PNA/DNA duplex stability. CD spectra of 6, 7, and 8 mer PNA/DNA duplexes showed similar signal, while 10, 12, and 16 mer duplexes did not. The average difference in Delta G degrees (37) for short PNA/DNA duplexes with identical nearest-neighbor pairs was only 3.5%, whereas that of longer duplexes (10, 12, and 16 mers) was 16.4%. Therefore, the nearest-neighbor model seems to be useful at least for the short PNA/DNA duplexes. Thermodynamics of PNA/DNA duplexes containing 1--3 bulge residues were also studied. While the stability of the 12 mer DNA/DNA duplex decreased as the number of bulge bases increases, the number of bulge bases in PNA/DNA unchanged the duplex stability. Thus, the influence of bulge insertion in the PNA/DNA duplexes is different from that of a DNA/DNA duplex. This might be due to the different base geometry in a helix which may potentially make hydrogen bonds in a base pair and stacking interaction unfavorable compared with DNA/DNA duplexes.     

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.     

Ability of polymerase eta and T7 DNA polymerase to bypass bulge structures

DNA misalignment occurs in homopolymer tracts during replication and can lead to frameshift mutations. Polymerase (pol) recognition of primer-templates containing bulge structures and the transmission of a bulge through a polymerase binding site or replication complex are important components of frameshift mutagenesis. In this report, we describe the interaction of the catalytic core of pol eta with primer-templates containing bulge structures by single round primer extension. We found that pol eta could stabilize a frayed primer terminus, which enhances its ability to extend primer-templates containing bulges. Based on methylphosphonate-DNA mapping, pol eta interacts with the single strand template but not appreciably with the template strand of the DNA stem greater than two nucleotides from the primer terminus. These latter characteristics, combined with the ability to stabilize a frayed primer terminus, may explain why primer-templates containing template bulges are extended so efficiently by pol eta. Although pol eta could accommodate large bulges and continue synthesis without obstruction, bulge structures in the template, but not in the primer, caused termination of the T7 DNA replication complex. Terminations occurred when the template bulge neared the helix-loop-helix domain of the polymerase thumb. Terminations were not observed, however, when bulge structures approached the site of interaction of the DNA with the extended thumb and thioredoxin. At low temperature, however, terminations did occur at this site.     

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype–phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction‐dependent destabilization of the underlying double helix, and a self‐structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self‐structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size‐dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1–12, 2014.     

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.