Hydrogen bonding contributes to the selectivity of nucleotide incorporation opposite an oxidized abasic lesion

The ability of DNA polymerases to maintain the integrity of the genome even after it has been structurally altered is vital. There is considerable interest in determining the structural properties of the DNA template that polymerases recognize when determining which nucleotide to add to a nascent strand. Mechanistic, synthetic, and structural chemistries have been used to study how DNA polymerase activity is affected by size, shape, pi-stacking, and hydrogen bonds of the template molecules. Herein, we probe the structural aspects of abasic lesions that result in their distinct coding potential in Escherichia coli despite lacking a Watson-Crick base. In particular, we investigate why bypass of 2-deoxyribonolactone (L) results in significant amounts of dG incorporation opposite the lesion, whereas other abasic lesions (e.g., AP) adhere to the "A-rule". Experiments using synthetic analogues reveal that DNA polymerase V bypasses L and increased levels of dG incorporation result from a hydrogen bonding interaction between the carbonyl oxygen and dG. These results show that a DNA polymerase utilizes hydrogen bonding as one structural parameter when decoding an abasic lesion.     

Incorporation of a minimal nucleotide into DNA

Abasic sites are amongst the most frequent DNA lesions and result from spontaneous hydrolysis of the glycosidic bond or from the removal of damaged nucleobases. These depurination events can also occur on free deoxyribonucleoside triphosphates present in cells and lead to the formation of an abasic site triphosphate of which very little is known. Herein, we report the synthesis and biochemical characterization of the minimal triphosphate dФTP. Unexpectedly, dФTP is tolerated by various DNA polymerases and the incorporation efficiency obeys the A-rule. Single incorporation of dФMP units were also observed opposite abasic sites and the addition of prosthetic molecules mimicking base-pairs do not seem to favor the process.     

Characterization and mechanism of formation of tandem lesions in DNA by a nucleobase peroxyl radical

5,6-Dihydro-2'-deoxyuridin-6-yl (1) was independently generated via photolysis of 3. The radical is an analogue of the major reactive species produced from thymidine upon reaction with hydroxyl radical, which is the dominant DNA-damaging agent produced by the indirect effect of gamma-radiolysis. Under aerobic conditions, the peroxyl radical (2) derived from 1 reacts approximately 82% of the time with either the 5'- or 3'-adjacent nucleotide to produce two contiguously damaged nucleotides, known as tandem lesions. The structures and distribution of tandem lesions were investigated using probes that selectively detect abasic sites, ESI-MS/MS, and competition kinetics. In addition to 2-deoxyribonolactone, nonoxidized abasic sites were detected. 18O-Labeling verified that H2O was the source of oxygen in the abasic sites, but that O2 was the source of the oxygen in the 5,6-dihydro-6-hydroxy-2'-deoxyuridine derived from 2. ESI-MS/MS experiments, in conjunction with isotopic labeling, identified several products and provided direct evidence for peroxyl radical addition to the adjacent thymine bases. Kinetic studies revealed that peroxyl radical addition to the 5'-thymine was favored by approximately 4-5-fold over C1'-hydrogen atom abstraction from the respective deoxyribose ring, and that 2-deoxyribonolactone formation accounts for approximately 11% of the total amount of tandem lesions produced. These results suggest that tandem lesions, whose biochemical effects are largely unknown, constitute a major family of DNA damage products produced by the indirect effect of gamma-radiolysis.     

Thermodynamic Impact of Abasic Sites on Simulated Translesion DNA Synthesis

Loss of a base in DNA and the creation of an abasic (apurinic/apyrimidinic, AP) site is a frequent lesion that may occur spontaneously, or as a consequence of the action of DNA‐damaging agents. The AP lesion is mutagenic or lethal if not repaired. We report a systematic thermodynamic investigation by differential scanning calorimetry on the evolution, during primer extension, of a model AP site in chemically simulated DNA translesion synthesis. Incorporation of dAMP (deoxyadenosine monophosphate), as well as dTMP (deoxythymidine monophosphate), opposite an AP site is enthalpically unfavorable, although incorporation of dTMP is more enthalpically unfavorable than that of dAMP. This finding is in a good agreement with experimental data showing that AP sites block various DNA polymerases of eukaryotic and prokaryotic origin and that, if bypassed, dAMP is preferentially inserted, whereas insertion of dTMP is less likely. The results emphasize the importance of thermodynamic contributions to the insertion of nucleotides opposite an AP site by DNA polymerases.     

Histone-catalyzed cleavage of nucleosomal DNA containing 2-deoxyribonolactone

Oxidized abasic sites such as 2-deoxyribonolactone (L) are produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor natural products. 2-Deoxyribonolactone is labile under alkaline conditions, but its half-life in free DNA at pH 7.5 is approximately 1 week. Independent generation of L at defined positions within nucleosomes reveals that the histone proteins catalyze strand scission and increase the rate between 11- and ～43-fold. Mechanistic studies indicate that DNA-protein cross-links are not intermediates en route to strand scission and that C2 deprotonation is the rate-determining step. The use of mutant histone H4 proteins demonstrates that the lysine-rich tail that is often post-translationally modified in cells contributes to the cleavage of L but is not the sole source of the enhanced cleavage rates. Consideration of DNA repair in cells suggests that L formation in nucleosomal DNA as part of bistranded lesions by antitumor antibiotics results in de facto double strand breaks, the most deleterious form of DNA damage.     

基于核酸脱碱基位点的铽离子荧光增强型单核苷酸多态性识别研究

基于核酸脱碱基(AP)位点构建了无机配体稀土铽离子(Tb^3＋)荧光增强型单核苷酸多态性(SNP)识别方法. 在目标链靶标碱基对应的探针链上相应位置引入AP位点, 发现Tb^3＋可以选择性地结合在AP位点, 光激发时发生从DNA碱基到结合的Tb^3＋的能量转移, 使Tb^3＋特征荧光显著增强. 这种荧光增强作用与靶标碱基及AP位点侧翼碱基类型密切相关. 当靶标碱基和侧翼碱基为G时, 荧光最强. 该方法可用于区分肿瘤抑制基因p53密码子177位的C/G碱基变异.     

Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as "rollamers". Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain ("rollamerization"). We demonstrate that such "rollameric" migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor "rollamers" with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic "universal hinge" relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.     

Evaluating the contributions of desolvation and base-stacking during translesion DNA synthesis

DNA polymerases catalyze the insertion of a nucleoside triphosphate into the growing polymer chain using the template strand as a guide. Numerous factors such as hydrogen bonding interactions, base-stacking contributions, and desolvation play important roles in controlling the efficiency and fidelity of this process. We previously demonstrated that 5-nitro-indolyl-2'-deoxyriboside triphosphate, a non-natural nucleobase with enhanced base-stacking properties, was more efficiently inserted opposite a non-templating DNA lesion compared to natural templating nucleobases (E. Z. Reineks and A. J. Berdis, Biochemistry, 2004, 43, 393-404). The catalytic enhancement was proposed to reflect increased base-stacking interactions of the non-natural nucleobase with the polymerase and DNA. However, the effects of desolvation could not be unambiguously refuted. To further address the contributions of base stacking and desolvation during translesion DNA replication, we synthesized indolyl-2'-deoxyriboside triphosphate, a nucleobase devoid of nitro groups, and measured its efficiency of enzymatic insertion into modified and unmodified DNA. Removal of the nitro group reduces the catalytic efficiency for insertion opposite an abasic site by 3600-fold. This results from a large decrease in the rate of polymerization (similar 450-fold) coupled with a modest decrease in binding affinity (similar 8-fold). Since both non-natural nucleobases show the same degree of hydrophobicity, we attribute this reduction to the loss of base-stacking contributions rather than desolvation capabilities. Indolyl-2'-deoxyriboside triphosphate can also be inserted opposite natural nucleobases. Surprisingly, the catalytic efficiency for insertion is nearly identical to that measured for insertion opposite an abasic site. These data are discussed within the context of pi-electron interactions of the incoming nucleobase with the polymerase:DNA complex. Despite this lack of insertion selectivity, the polymerase is unable to extend beyond the non-natural nucleobase. This result indicates that indolyl-2'-deoxyriboside triphosphate acts as an indiscriminate chain terminator of DNA synthesis that may have unique therapeutic applications.     

Independent generation and characterization of a C2'-oxidized abasic site in chemically synthesized oligonucleotides

Abasic lesions, which are formed endogenously and as a consequence of exogenous agents, are lethal and mutagenic. Hydrogen atom abstraction from C2' in DNA under aerobic conditions produces an oxidized abasic lesion (C2-AP), along with other forms of DNA damage. The effects of C2-AP on DNA structure and function are not well understood. A method for the solid-phase synthesis of oligonucleotides containing C2-AP lesions is reported. The lesion is released via periodate oxidation of a triol containing a vicinal diol. The triol is introduced via a phosphoramidite that is compatible with standard oligonucleotide synthesis and deprotection conditions. UV-melting studies indicate that the C2-AP lesion has a comparable effect on the thermal stability of duplex DNA as other abasic lesions. The C2-AP lesion is rapidly cleaved by piperidine at 90 degrees C. However, cleavage by NaOH (0.1 M, 37 degrees C) shows that C2-AP is considerably less labile (t(1/2) = 3.3 +/- 0.2 h) than other abasic lesions.     

Selective detection of 2-deoxyribonolactone in DNA

2-Deoxyribonolactone (L) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents. It exhibits unique biological effects with respect to its proclivity to form DNA-protein cross-links and promutagenic base pairs. Recent evidence suggests that the levels of this lesion caused by oxidative stress are underestimated. We have developed a simple, selective method for detecting subpicomole amounts of L in DNA. The method takes advantage of the selective reaction of the butenolide (2) derived from beta-elimination from L with a biotinylated derivative of cysteine. This method will be useful for analyzing the levels of this oxidized abasic site in DNA.     

Molecular modeling study of DNA abasic sites

We use molecular modeling calculations to study the structure and the flexibility of abasic (AP sites) and for the design of anticancer drugs targeted against AP sites. For either adenine or cytosine on the opposing strand within the same sequence context, the results are in line with experimental data which show that the two unpaired bases lead to intrahelical forms, but with differences in induced curvature. Results on flexibility, indicate that the two duplexes have the same bending rigidity for cytosine. In previous work a series of polyfunctional molecules, such as ATAc, were designed to selectively recognize and cleave abasic sites in DNA. The nitrobenzamide group which was added to the ATAc molecule to obtain a new molecule, termed ATAc4, can induce a second lesion under irradiation in close proximity to the abasic site. The different conformations of ATAc4 interacting with a DNA oligomer containing a stable analog of the abasic site were compared to the photoinduced cleavage pattern observed experimentally. Received: 16 September 1999 / Accepted: 3 February 2000 / Published online: 12 May 2000     

Optimization of non-natural nucleotides for selective incorporation opposite damaged DNA

The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the pi-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of shape and pi-electron density. In this report, we describe the synthesis and kinetic characterization of four novel nucleotide analogs, 5-cyanoindolyl-2'-deoxyriboside 5'-triphosphate (5-CyITP), 5-ethyleneindolyl-2'-deoxyriboside 5'-triphosphate (5-EyITP), 5-methylindolyl-2'-deoxyriboside 5'-triphosphate (5-MeITP), and 5-ethylindolyl-2'-deoxyriboside 5'-triphosphate (5-EtITP). Kinetic analyses indicate that the overall catalytic efficiencies of all four nucleotides are related to their base-stacking properties. In fact, the catalytic efficiency for nucleotide incorporation opposite an abasic site displays a parabolic trend in the overall pi-electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA approximately 100-fold worse than opposite an abasic site. These data indicate that selectivity for incorporation opposite damaged DNA can be achieved through optimization of the base-stacking properties of the incoming nucleotide.     

Sequence dependence of cytochrome c electrochemistry on DNA modified electrodes: Effect of hydrogen bonding of a ligand to nucleobases opposite an abasic site

The binding of a hydrogen bond-forming ligand to abasic (AP) site-containing DNA was electrochemically investigated for discrimination of a nucleotide opposite the AP site. The surface of a gold electrode was modified by AP site-containing DNA duplexes on which cytochrome c (Cyto c) was attached electrostatically as a probe. Cyto c showed quasi-reversible electrochemical behavior depending on the base opposite the AP site. When the base opposite the AP site was cytosine, much slower kinetics of Cyto c electron transfer was observed. This observation could be explained by previous reports that the base stacking was disturbed to a much greater extent because the cytosine base opposite the AP site was flipped out extra-helically. The binding of a hydrogen bond-forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), to cytosine opposite the AP site could significantly improve the electrochemical behavior of Cyto c, indicating effective base stacking due to the AMND binding. The present method demonstrates an easy way for investigating the binding of a small ligand to the AP site through DNA-mediated charge transfer.     

Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6-4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41-mer DNA containing a (6-4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases alpha, beta and delta (pol alpha, pol beta and pol delta). None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration. When a single-chain Fv (scFv) with high affinity for the (6-4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-pyrimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively. These results suggest that the scFv can bind to the template DNA containing a (6-4) photoproduct and inhibit extension reactions by polymerases.     

Wavelength dependence of ultraviolet-induced DNA damage distribution: involvement of direct or indirect mechanisms and possible artefacts.

DNA damage profiles have been established in plasmid DNA using purified DNA repair enzymes and a plasmid relaxation assay, following exposure to UVC, UVB, UVA or simulated sunlight (SSL). Cyclobutane pyrimidine dimers (CPDs) are revealed as T4 endonuclease V-sensitive sites, oxidation products at purine and pyrimidine as Fpg- and Nth-sensitive sites, and abasic sites are detected by Nfo protein from Escherichia coli. CPDs are readily detected after UVA exposure, though produced 10(3) and 10(5) times less efficiently than by UVB or UVC, respectively. We demonstrate that CPDs are induced by UVA radiation and not by contaminating UVB wavelengths. Furthermore, they are produced at doses compatible with human exposure and are likely to contribute to the mutagenic specificity of UVA [E. Sage et al., Proc. Natl. Acad. Sci. USA 93 (1996) 176-180]. Oxidative damage is induced with a linear dose dependence, for each region of the solar spectrum, with the exception of oxidized pyrimidine and abasic sites, which are not detectable after UVB irradiation. The distribution of the different classes of photolesions varies markedly, depending on wavelengths. However, the unexpectedly high yield of oxidative lesions, as compared to CPDs, by UVA and SSL led us to investigate their production mechanism. An artificial formation of hydroxyl radicals is observed, which depends on the material of the sample holder used for UVA irradiation and is specific for long UV wavelengths. Our study sheds light on a possible artefact in the production of oxidative damage by UVA radiation. Meanwhile, after eliminating some potential sources of the artefact ratios of CPDs to oxidized purine of three and five upon irradiation with UVA and SSL, respectively, are still observed, whereas these ratios are about 140 and 200 after UVC and UVB irradiation.     

Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize DNA Duplexes Containing O6‐Alkylguanine Adducts

Chemical alkylation of DNA produces potentially toxic and mutagenic damage such as O⁶‐alkylguanine (O⁶‐alkylG) adducts. Non‐natural nucleoside analogues that pair with DNA adducts provide a potential basis for studying damaged DNA. Herein, we evaluated the base pairing properties of elongated nucleoside analogues containing napthalene‐derived tricyclic nucleobases as DNA adduct‐pairing nucleoside analogues in DNA hybridization probes. DNA duplex melting studies revealed that the elongated nucleoside analogs formed more stable base pairs opposite O⁶‐alkylG than G and were better able to distinguish between G, O⁶‐alkylG, and an abasic site than any previously described nucleoside analogue. DNA duplexes containing an elongated base analogue exhibited different fluorescence intensities when paired opposite O⁶‐alkylG vs. G or abasic sites. Their selectivity for stabilizing alkylated DNA make the elongated hydrophobic base analogues improved candidates for incorporating into DNA hybridization probes targeting O⁶‐alkylG.     

Oxidative DNA damage following photoexcitation of daunomycin: Direct role of oxygen

The chemistry of the photoactivation of daunomycin–DNA complexes is reported and the mechanism is elucidated. We quantitatively assessed the type of DNA damage, such as strand breaks, oxidized bases, and abasic sites, that arise using a plasmid relaxation assay coupled with DNA repair endonucleases. Photoexcitation of daunomycin leads to oxidative DNA damage in a dose- and irradiation time-dependent manner and guanine-specific oxidized purines are substantially produced under these conditions. Oxidative DNA base damage was also inhibited by argon degassing, indicating that guanine-specific damage arises from an oxygen-dependent mechanism. In addition, photoexcitation of daunomycin–DNA complexes leads to superoxide anion radical formation. From these studies of the actual product formed, we conclude that a charge transfer is a main driving force of the mechanism.     

Synthesis of abasic locked nucleic acid and two seco-LNA derivatives and evaluation of their hybridization properties compared with their more flexible DNA counterparts

To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid) three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1'-O4' bond of an LNA monomer or complete removal of the O4'-furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

DNA-duplexes containing abasic sites: correlation between thermostability and acoustic wave properties

Aldehydic apurinic or apyrimidinic sites that lack a nucleobase moiety are one of the most common forms of toxic lesions in DNA. In the present study, a close structural analog of such a site, the 2-(hydroxymethyl) tetrahydrofuranyl residue, was synthesized in order to act as a model for damaged nucleic acid probes. Prepared oligodeoxyribonucleotides containing one, two or three abasic sites were hybridized to complementary sequences immobilized on a gold surface using the neutravidin-biotin interaction for study by thickness shear mode acoustic wave detector. Measurement of the complex electrical impedance of an AT-cut quartz device with immobilized biotinylated nucleotide allowed the detection of changes of series resonance frequency, Deltafs, and motional resistance, Rm, associated with duplex formation. The changes as detected by the acoustic wave method correlated well with the thermostability of DNA duplexes in solution. With respect to the latter, UV-monitored melting curves indicate that both the number of sites and their localization in the double-stranded structure influence the amount by which a 19 b.p. duplex is destabilized. The presence of 3 abasic sites completely destabilized the DNA duplex.