Abasic Site In Duplex DNA Research Articles

Novel Processes Associated with the Repair of Interstrand Cross-Links Derived from Abasic Sites in Duplex DNA: Roles for the Base Excision Repair Glycosylase NEIL3 and the SRAP Protein HMCES.

Recent studies have defined a novel pathway for the repair of interstrand cross-links derived from the reaction of an adenine residue with an apurinic/apyrimidinic (AP) site on the opposing strand of DNA (dA-AP ICL). Stalling of a replication fork at the dA-AP ICL triggers TRAIP-dependent ubiquitylation of the CMG helicase that recruits the base excision repair glycosylase NEIL3 to the lesion. NEIL3 unhooks the dA-AP ICL to regenerate the native adenine residue on one strand and an AP site on the other strand. Covalent capture of the abasic site by the SRAP protein HMCES protects against genomic instability that would result from cleavage of the abasic site in the context of single-stranded DNA at the replication fork. After repair synthesis moves the HMCES-AP adduct into the context of double-stranded DNA, the DNA-protein cross-link is resolved by a nonproteolytic mechanism involving dissociation of thiazolidine attachment. The AP site in duplex DNA is then repaired by the base excision repair pathway.

Interstrand Cross-Link Formation Involving Reaction of a Mispaired Cytosine Residue with an Abasic Site in Duplex DNA.

The formation of interstrand cross-links in duplex DNA is important in biology, medicine, and biotechnology. Interstrand cross-links arising from the reaction of the aldehyde residue of an abasic (apurinic or AP) site with the exocyclic amino groups of guanine or adenine residues on the opposing strand of duplex DNA have previously been characterized. The canonical nucleobase cytosine has an exocyclic amino group but its ability to form interstrand cross-links by reaction with an AP site has not been characterized before now. Here it is shown that substantial yields of interstrand cross-links are generated in sequences having a mispaired cytosine residue located one nucleotide to the 3'-side of the AP site on the opposing strand (e.g., 5'XA/5'CA, where X = AP). Formation of the dC-AP cross-link is pH-dependent, with significantly higher yields at pH 5 than pH 7. Once formed, the dC-AP cross-link is quite stable, showing less than 5% dissociation over the course of 96 h at pH 7 and 37 °C. No significant yields of cross-link are observed when the cytosine residue is paired with its Watson-Crick partner guanine. It was also shown that a single AP site can engage with multiple nucleobase cross-linking partners in some sequences. Specifically, the dG-AP and dC-AP cross-links coexist in dynamic equilibrium in the sequence 5'CXA/5'CAG (X = AP). In this sequence, the dC-AP cross-link dominates. However, in the presence of NaBH3CN, irreversible reduction of small amounts of the dG-AP cross-link present in the mixture shifts the equilibria away from the dC-AP cross-link toward good yields of the dG-APred cross-link.

Nitroxide-Derived N-Oxide Phenazines for Noncovalent Spin-Labeling of DNA.

Two o-benzoquinone derivatives of isoindoline were synthesized for use as building blocks to incorporate isoindoline nitroxides into different compounds and materials. These o-quinones were condensed with a number of o-phenylenediamines to form isoindoline-phenazines in high yields. Subsequent oxidation gave phenazine-di-N-oxide isoindoline nitroxides that were evaluated for noncovalent and site-directed spin-labeling of duplex DNA and RNA that contained abasic sites. Although only minor binding was observed for RNA, the unsubstituted phenazine-N,N-dioxide tetramethyl isoindoline nitroxide showed high binding affinity and selectivity towards abasic sites in duplex DNA that contained cytosine as the orphan base.

Surface-enhanced Raman spectroscopy (SERS) characterisation of abasic sites in DNA duplexes.

In this study, direct surface-enhanced Raman scattering (SERS) spectroscopy is used as an exquisite nano-optical tool for ultrasensitive structural characterisation of abasic sites in DNA. In addition, the conformational discrimination (intra- vs. extra-helical) of the nucleobase opposite to the abasic site was also achieved.

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3’ddR5p) at the 3’-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3’ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3’ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems.

Simple, High-Yield Syntheses of DNA Duplexes Containing Interstrand DNA-DNA Cross-Links Between an N(4) -Aminocytidine Residue and an Abasic Site.

The protocol describes the preparation and purification of interstrand DNA-DNA cross-links derived from the reaction of an N(4) -aminocytidine residue with an abasic site in duplex DNA. The procedures employ inexpensive, commercially available chemicals and enzymes to carry out post-synthetic modification of commercially available oligodeoxynucleotides. The yield of cross-linked duplex is typically better than 90%. If purification is required, the cross-linked duplex can be readily separated from single-stranded DNA starting materials by denaturing gel electrophoresis. The resulting covalent hydrazone-based cross-links are stable under physiologically relevant conditions and may be useful for biophysical studies, structural analyses, DNA repair studies, and materials science applications. © 2016 by John Wiley & Sons, Inc.

Crystal structure of a nucleoside model for the inter-strand cross-link formed by the reaction of 2'-de-oxy-guanosine and an abasic site in duplex DNA.

The title compound, 9-[(2R,4S,5R)-4-hy-droxy-5-(hy-droxy-meth-yl)tetra-hydro-furan-2-yl]-2-{[(2R,4S,5R)-4-meth-oxy-5-(meth-oxy-meth-yl)tetra-hydro-furan-2-yl]amino}-1H-purin-6(9H)-one, C17H25N5O7, crystallizes with two independent mol-ecules (A and B) in the asymmetric unit. In the crystal, the guanosine moieties of mol-ecules A and B are linked by N-H⋯N and O-H⋯N hydrogen-bonding inter-actions, forming ribbons which are stacked to form columns along [100]. These columns are then linked by O-H⋯O hydrogen bonds between the ribose moieties and numerous C-H⋯O inter-actions to complete the three-dimensional structure.

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N 2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.

A Simple, High‐Yield Synthesis of DNA Duplexes Containing a Covalent, Thermally Cleavable Interstrand Cross‐Link at a Defined Location

AbstractInterstrand DNA–DNA cross‐links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross‐links are important, but not well understood. The preparation of chemically well‐defined cross‐linked DNA substrates represents a significant challenge in the study of cross‐link repair. Here a simple method is reported that employs “post‐synthetic” modifications of commercially available 2′‐deoxyoligonucleotides to install a single cross‐link in high yield at a specified location within a DNA duplex. The cross‐linking process exploits the formation of a hydrazone between a non‐natural N4‐amino‐2′‐deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross‐link is stable under physiological conditions, but can be readily dissociated and re‐formed through heating–cooling cycles.

A simple, high-yield synthesis of DNA duplexes containing a covalent, thermally cleavable interstrand cross-link at a defined location.

Interstrand DNA-DNA cross-links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross-links are important, but not well understood. The preparation of chemically well-defined cross-linked DNA substrates represents a significant challenge in the study of cross-link repair. Here a simple method is reported that employs "post-synthetic" modifications of commercially available 2'-deoxyoligonucleotides to install a single cross-link in high yield at a specified location within a DNA duplex. The cross-linking process exploits the formation of a hydrazone between a non-natural N(4) -amino-2'-deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross-link is stable under physiological conditions, but can be readily dissociated and re-formed through heating-cooling cycles.

Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA.

A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link "remnant" released by enzymatic digestion of DNA duplexes containing the dG-AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N(2)-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-AP cross-link, respectively. These results establish the chemical connectivity of the dG-AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG-AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

Base excision repair enzymes protect abasic sites in duplex DNA from interstrand cross-links.

Hydrolysis of the N-glycosyl bond between a nucleobase and deoxyribose leaves an abasic site within duplex DNA. The abasic site can react with exocyclic amines of nucleobases on the complementary strand to form interstrand DNA-DNA cross-links (ICLs). We find that several enzymes from the base excision repair (BER) pathway protect an abasic site on one strand of a DNA duplex from cross-linking with an amine on the opposing strand. Human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) accomplish this by binding tightly to the abasic site and sequestering it. AAG protects an abasic site opposite T, the product of its canonical glycosylase reaction, by a factor of ∼10-fold, as estimated from its inhibition of the reaction of an exogenous amine with the damaged DNA. Human apurinic/apyrimidinic site endonuclease 1 and E. coli endonuclease III both decrease the amount of ICL at equilibrium by generating a single-strand DNA nick at the abasic position as it is liberated from the cross-link. The reversibility of the reaction between amines and abasic sites allows BER enzymes to counter the potentially disruptive effects of this type of cross-link on DNA transactions.

Interstrand DNA-DNA cross-link formation between adenine residues and abasic sites in duplex DNA.

The loss of a coding nucleobase from the structure of DNA is a common event that generates an abasic (Ap) site (1). Ap sites exist as an equilibrating mixture of a cyclic hemiacetal and a ring-opened aldehyde. Aldehydes are electrophilic functional groups that can form covalent adducts with nucleophilic sites in DNA. Thus, Ap sites present a potentially reactive aldehyde as part of the internal structure of DNA. Here we report evidence that the aldehyde group of Ap sites in duplex DNA can form a covalent adduct with the N6-amino group of adenine residues on the opposing strand. The resulting interstrand DNA–DNA cross-link occurs at 5′-ApT/5′-AA sequences in remarkably high yields (15–70%) under physiologically relevant conditions. This naturally occurring DNA-templated reaction has the potential to generate cross-links in the genetic material of living cells.

Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline.

A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 μM. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.

DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences

The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.

Effect of N3 Modifications on the Affinity of Spin Label ç for Abasic Sites in Duplex DNA

Noncovalent site-directed spin labeling (NC-SDSL) of abasic sites in duplex DNAs with the spin label ç, a cytosine analogue, is a promising approach for spin-labeling nucleic acids for EPR spectroscopy. In an attempt to increase the affinity of ç for abasic sites, several N3 derivatives were prepared, and their binding affinities were determined by EPR spectroscopy. Most of the N3 substituents had a detrimental effect on binding. The triazole-linked polyethylene-glycol derivative (12 a) showed a 15-fold decrease in affinity, whereas the binding affinities of ethyl azido (8 b) and hydroxyl (8 c) derivatives were five- to sixfold lower. The spin-labeled nucleoside Ç showed only a twofold decrease, thus binding better than 8 c, even though it contains the larger 2'-deoxyribose substituent at N3 instead of a 2-hydroxyethyl group. N3 derivatives that contained the basic ethyl amino (9) or ethyl guanidino (10) substituents had both higher binding affinity and solubility, attributed to their cationic charge at neutral pH. Compounds 9 and 10 are promising candidates for NC-SDSL of nucleic acids, for distance measurements by pulsed EPR spectroscopy.

Base Pairing at the Abasic Site in DNA Duplexes and Its Application in Adenosine Aptasensors

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 μM for adenosine and 41.8 μM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 μM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.

Highly selective binding of naphthyridine with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes

We report on highly selective binding of a naphthyridine derivative with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes; the binding-induced fluorescence quenching is applicable to the analysis of a C-related single-base mutation in DNAs amplified by PCR.

Structural changes of an abasic site in duplex DNA affect noncovalent binding of the spin label ç

The influence of structural changes of an abasic site in duplex DNA on noncovalent and site-directed spin labeling (NC-SDSL) of the spin label ç were examined with electron paramagnetic resonance (EPR) spectroscopy. The binding affinities of ç to sixteen different DNA duplexes containing all possible sequences immediately flanking the abasic site were determined and the results showed that the binding of ç is highly flanking-sequence dependent. In general, a 5′-dG nucleotide favors the binding of the spin label. In particular, 5′-d(G__T) was the best binding sequence whereas 5′-d(C__T) showed the lowest affinity. Changing the structure of the abasic site linker from a tetrahydrofuran analog (F) to the anucleosidic C3-spacer (C3) does not appreciably affect the binding of ç to the abasic site. For efficient binding of ç, the abasic site needs to be located at least four base pairs away from the duplex end. Introducing a methyl substituent at N3 of ç did not change the binding affinity, but a decreased binding was observed for both N3-ethyl and -propyl groups. These results will guide the design of abasic site receptors and spin label ligands for NC-SDSL of nucleic acids.

Nitroxides and nucleic acids: Chemistry and electron paramagnetic resonance (EPR) spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy has increasingly been applied for the study of nucleic acid structure and dynamics. Such studies require incorporation of free radicals (spin labels) into the biopolymer. The labels can be incorporated during chemical synthesis of the oligomer (phosphoramidite approach) or postsynthetically, by reaction of a spin-labeling reagent with a reactive functional group on the oligonucleotide. Incorporation of the rigid nitroxide spin label Ç is an example of the phosphoramidite method, and reaction of a spin-labeled azide with an alkyne-modified oligomer to yield a triazole-derived, spin-labeled nucleotide illustrates the postsynthetic spin-labeling strategy. Characterization and application of these labels to study structural features of DNA by EPR spectroscopy is discussed. Finally, a new spin-labeling strategy is described for nucleic acids that relies on noncovalent interactions between a spin-labeled nucleobase and an abasic site in duplex DNA.