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Preparation and analysis of oligonucleotides containing the c4'-oxidized abasic site and related mechanistic probes.

ABSTRACT: The C4'-oxidized abasic site (C4-AP) is produced by a variety of DNA damaging agents. This alkali labile lesion can exist in up to four diastereomeric cyclic forms, in addition to the acyclic keto-aldehyde. Synthetic oligonucleotides containing the lesion were prepared from a stable photochemical precursor. Chemical integrity of the lesion containing oligonucleotides was probed using phosphodiesterase lability. Analysis of the 3',5'-phosphate diester of the monomeric lesion released from single diastereomers of photolabile precursors by 1H NMR indicates that isomerization of the hemiacetal and/or hemiketal is rapid. The syntheses and characterization of oligonucleotides containing configurationally stable analogues of C4-AP, which serve as mechanistic probes for deciphering the structural basis of the biochemical and biological effects of the C4'-oxidized abasic lesion, are also described.

SUBMITTER: Kim J

PROVIDER: S-EPMC1382185 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Preparation and analysis of oligonucleotides containing the c4'-oxidized abasic site and related mechanistic probes.

Kim Jaeseung J Kreller Cortney R CR Greenberg Marc M MM

The Journal of organic chemistry 20050901 20

The C4'-oxidized abasic site (C4-AP) is produced by a variety of DNA damaging agents. This alkali labile lesion can exist in up to four diastereomeric cyclic forms, in addition to the acyclic keto-aldehyde. Synthetic oligonucleotides containing the lesion were prepared from a stable photochemical precursor. Chemical integrity of the lesion containing oligonucleotides was probed using phosphodiesterase lability. Analysis of the 3',5'-phosphate diester of the monomeric lesion released from single ...[more]

PMID: 16277338

Similar Datasets

Synthesis and analysis of oligonucleotides containing abasic site analogues.

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Scope and mechanism of interstrand cross-link formation by the C4'-oxidized abasic site.

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Long patch base excision repair compensates for DNA polymerase ? inactivation by the C4'-oxidized abasic site.

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Synthesis of cross-linked DNA containing oxidized abasic site analogues.

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| S-EPMC4084848 | biostudies-other

Histone modification via rapid cleavage of C4'-oxidized abasic sites in nucleosome core particles.

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| S-EPMC3638250 | biostudies-literature

The A-Rule and Deletion Formation During Abasic and Oxidized Abasic Site Bypass by DNA Polymerase ?.

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| S-EPMC5499511 | biostudies-literature

Tandem mass spectrometry-based detection of c4'-oxidized abasic sites at specific positions in DNA fragments.

Project description:Oxidative damage to DNA has been linked to aging, cancer, and other biological processes. Reactive oxygen species and various antitumor agents including bleomycin and ionizing radiation have been shown to cause oxidative DNA sugar damage. Detection of DNA lesions is important for understanding the toxicological or therapeutic consequences associated with such agents. C4'-oxidized abasic sites (C4-AP) are produced by the antitumor drug bleomycin and ionizing radiation. The currently available methods for the detection of C4-AP cannot provide both structural and sequence information. We have developed an LC-ESI-MS-based approach for specific detection and mapping of C4-AP from a mixture of lesions. We show using Fe-bleomycin-damaged DNA that C4-AP can be detected at cytosine and thymine sites by direct MS analysis. Our results reveal that collision-induced dissociation of C4-AP-containing oligonucleotides results in preferential fragmentation at C4-AP sites with the formation of the unique a* ions (18 amu more than the a-B ions) that allow mapping of the C4-AP sites. Various chemical modification strategies (e.g., reduction with NaBH4 and NaBD4 and derivatization with methoxyamine and hydrazine, followed by LC-MS analysis) were also used for unambiguous detection of C4-AP sites. Finally, we show that the methods described here can detect the presence of C4-AP at specific sites in a complex sample such as hydroxyl radical-damaged DNA. The LC-MS approach was also used for the simultaneous detection of the other C4'-oxidation end product, 3'-phosphoglycolate, at a specific site in hydroxyl radical-damaged DNA. Thus, LC-MS provides a rapid and direct approach for the detection and mapping of oxidative DNA lesions.

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DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics.

Project description:Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5'-CCA AAG6 XA8C CGG G-3', where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites. Biochemistry, 46, 3096-3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3' to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.

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Inhibition of short patch and long patch base excision repair by an oxidized abasic site.

Project description:5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents, including several antitumor antibiotics. DOB efficiently and irreversibly inhibits DNA polymerase ?, an essential base excision repair enzyme in mammalian cells. The generality of this mode of inhibition by DOB is supported by the inactivation of DNA polymerase ?, which may serve as a possible backup for DNA polymerase ? during abasic site repair. Protein digests suggest that Lys72 and Lys84, which are present in the lyase active site of DNA polymerase ?, are modified by DOB. Monoaldehyde analogues of DOB substantiate the importance of the 1,4-dicarbonyl component of DOB for efficient inactivation of Pol ? and the contribution of a freely diffusible electrophile liberated from the inhibitor by the enzyme. Inhibition of DNA polymerase ?'s lyase function is accompanied by inactivation of its DNA polymerase activity as well, which prevents long patch base excision repair of DOB. Overall, DOB is highly refractory to short patch and long patch base excision repair. Its recalcitrance to succumb to repair suggests that DOB is a significant source of the cytotoxicity of DNA damaging agents that produce it.

| S-EPMC2976803 | biostudies-literature

Preparation and analysis of oligonucleotides containing lesions resulting from C5'-oxidation.

Project description:[reaction: see text] Hydrogen atom abstraction from the C5'-position of nucleotides in DNA results in direct strand scission. The newly formed 5'-termini of the cleaved DNA consists of alkali-labile fragments of the oxidized nucleotide. One terminus contains a 5'-aldehyde as part of an otherwise undamaged nucleotide (T-al). A second more structurally distinct product that is produced in lower yields results from cleavage of the C4'-C5' carbon-carbon bond. The 1,4-dioxo-2-phosphorylbutane (DOB) is a precursor of the alkylating agent but-2-ene-1,4-dial. To facilitate studies on these lesions, methods for synthesizing oligodeoxynucleotides containing DOB or T-al at their 5'-termini were developed. The effects of these lesions on the UV-melting temperatures of duplex DNA, and their cleavage labilities were determined. T-al cleaves very slowly (t(1/2) = 100.7 h), whereas DOB has a half-life at 37 degrees C (pH 7.2) of less than 11 h. In addition, DOB forms a stable adduct very efficiently with Tris, which protects the abasic site against cleavage.

| S-EPMC1657062 | biostudies-literature

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