Preparation and Analysis of Oligonucleotides Containing Lesions Resulting from C5‘-Oxidation

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 (t1/2 = 100.7 h), whereas DOB has a half-life at 37 °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

Hole Migration is the Major Pathway Involved in Alkali-Labile Lesion Formation in DNA by the Direct Effect of Ionizing Radiation

Electron transfer via nucleobase hole migration has been extensively studied in recent years. Holes are produced in DNA via the direct effect of γ-radiolysis. However, the contribution of this pathway to DNA damage is difficult to discern because common products are formed via the direct and indirect (e.g., hydroxyl radical) pathways. A molecular probe (1) was designed that serves as a repository for holes in DNA. Upon one-electron oxidation, 1 fragments to fdU, which is an alkali-labile lesion. Incorporation of 1 in synthetic DNA enables one to determine the contribution of hole transfer to DNA damage by γ-radiolysis. We find that hole migration is the predominant pathway for alkali-labile lesion formation when DNA is exposed to 137Cs in solution

Irreversible Inhibition of DNA Polymerase β by an Oxidized Abasic Lesion

Irreversible Inhibition of DNA Polymerase β by an Oxidized Abasic Lesio

γ-Radiolysis and Hydroxyl Radical Produce Interstrand Cross-Links in DNA Involving Thymidine

Interstrand cross-links are minor components of the collection of products formed in DNA by ionizing radiation. Through their formation by other damaging agents, it is known that interstrand cross-links exert significant effects on replication and transcription. The structures of DNA interstrand cross-links produced as a result of γ-radiolysis are unknown. Using synthetic duplexes we found that interstrand cross-link formation required thymidine and occurred with G values of ∼10−4 nmol J−1. Enzymatic digestion of a tritiated substrate indicated that interstrand cross-links were derived from the reaction of 5-(2′-deoxyuridinyl)methyl radical (1) with the opposing 2′-deoxyadenosine to yield 5, which was identical to the product previously characterized when 1 was independently generated from a synthetic precursor. Conservative estimates indicated that 5 accounted for at least one-fourth of the interstrand cross-links produced in DNA by γ-radiolysis. Utilization of a probe designed specifically to detect hole migration suggested that ∼20% of the interstrand cross-links were produced by γ-radiolysis via this pathway. Experiments using an independent source of hydroxyl radical indicated that cross-links were also produced by this species. Hence, DNA interstrand cross-links arising from 1 should result from a variety of oxidative stress mechanisms

DNA Interstrand Cross-Link Formation by the 1,4-Dioxobutane Abasic Lesion

The oxidized abasic lesion 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) is produced concomitantly with a single-strand break by a variety of DNA-damaging agents that abstract a hydrogen atom from the C5′-position. Independent generation of the DOB lesion in DNA reveals that it reversibly forms interstrand cross-links (ICLs) selectively with a dA opposite the 3′-adjacent nucleotide. Product studies and the use of monoaldehyde models suggest that ICL formation involves condensation of the dialdehyde with the exocyclic amine. Mechanistic studies and inspection of molecular models indicate that the local DNA environment and proximity of the exocyclic amine determine the selectivity for reaction with dA. Proximity control of the electrophile’s reactivity is distinct from that of structurally similar freely diffusing molecules. ICL formation by a DOB lesion that is adjacent to a single-strand break is potentially significant because the product constitutes a “clustered” or “complex” lesion. Clustered lesions can lead to highly deleterious double-strand breaks upon nucleotide excision repair

Facile Quantification of Lesions Derived from 2‘-Deoxyguanosine in DNA

OxodG and Fapy•dG are two frequently formed DNA lesions that affect replication in vitro and in cells. They are also potentially important biomarkers for determining the effects of oxidative stress and aging in cells. We report the first method that enables one to selectively detect and individually quantify Fapy•dG and OxodG in DNA. The method relies upon selective chemical trapping of oxidized forms of the lesions with a biotinylated derivative of spermine. Selectivity for OxodG over Fapy•dG is achieved by varying the oxidant. The covalently tagged lesions are quantified using a fluorescence assay that is carried out in microtiter plates. The fluorescence assay is generally applicable to quantifying DNA lesions that can be tagged with biotin

Hydrogen Bonding Contributes to the Selectivity of Nucleotide Incorporation Opposite an Oxidized Abasic Lesion

Hydrogen Bonding Contributes to the Selectivity of Nucleotide Incorporation Opposite an Oxidized Abasic Lesio

Nucleotide Excision Repair of Chemically Stabilized Analogues of DNA Interstrand Cross-Links Produced from Oxidized Abasic Sites

Nucleotide excision repair is a primary pathway in cells for coping with DNA interstrand cross-links (ICLs). Recently, C4′-oxidized (C4-AP) and C5′-oxidized abasic sites (DOB) that are produced following hydrogen atom abstraction from the DNA backbone were found to produce ICLs. Because some of the ICLs derived from C4-AP and DOB are too unstable to characterize in biochemical processes, chemically stable analogues were synthesized [Ghosh, S., and Greenberg, M. M. (2014) <i>J. Org. Chem.</i> <i>79</i>, 5948–5957]. UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB was examined. The incision pattern for the ICL related to the C4′-oxidized abasic site was typical for UvrABC substrates. UvrABC cleaved both strands of the substrate containing the C4-AP ICL analogue, but it was a poor substrate. UvrABC incised <30% of the C4-AP ICL analogue over an 8 h period, raising the possibility that this cross-link will be inefficiently repaired in cells. Furthermore, double-strand breaks were not detected upon incision of an internally labeled hairpin substrate containing the C4-AP ICL analogue. UvrABC incised the stabilized analogue of the DOB ICL more efficiently (∼20% in 1 h). Furthermore, the incision pattern was unique, and the cross-linked substrate was converted into a single product, a double-strand break. The template strand was exclusively incised on the template strand on the 3′-side of the cross-linked dA. Although the outcomes of the interaction between UvrABC and these two cross-linked substrates are different from one another, they provide additional examples of how seemingly simple lesions (C4-AP and DOB) can potentially exert significant deleterious effects on biochemical processes

Preparation and Analysis of Oligonucleotides Containing Lesions Resulting from C5‘-Oxidation

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 (t1/2 = 100.7 h), whereas DOB has a half-life at 37 °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

Improved Utility of Photolabile Solid Phase Synthesis Supports for the Synthesis of Oligonucleotides Containing 3‘-Hydroxyl Termini

Oligonucleotides are synthesized on, and cleaved from, a solid phase support (6) using the o-nitrobenzyl intramolecular photochemical redox reaction. The yields of isolated oligonucleotides relative to yields obtained using conventional hydrolytic cleavage vary between 67% and 82.5%. Synthesis of oligonucleotides using phosphoramidites that do not contain N-benzoyl protecting groups enables one to photolytically cleave the biopolymers in good yields using a commonly available UV irradiation source. Tritium labeling indicates that less than 3% thymidine·thymidine photodimers are formed during photolytic cleavage of polythymidylates from 6 using a transilluminator. No UV-induced damage is detected via HPLC analysis of enzymatically digested oligonucleotides that were obtained following photolytic cleavage from 6