Synthesis and Characterization of Oligonucleotides Containing the C4′‐Oxidized Abasic Site Produced by Bleomycin and Other DNA Damaging Agents

Kim, Jaeseung; Gil, José María Mengual; Greenberg, Marc M.

doi:10.1002/ange.200352102

Cited by 18 publications

(38 citation statements)

References 35 publications

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“…To obtain an oligonucleotide containing a 2-deoxyribose (LysA30-AP), LysA30-U was treated with an excess amount of uracil DNA glycosylase as described previously (35). LysA30-L contained a 2-deoxyribonolactone (L) and was prepared by UV irradiation (350 nm) of parental oligonucleotide containing the appropriate photochemical precursors (36,37). Figure 1 shows the structures of these various abasic sites.…”

Section: Methodsmentioning

confidence: 99%

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

Kow

Bao²,

Minesinger³

et al. 2005

Nucleic Acids Research

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2-Deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polζ (rev3 mutant), but were only marginally reduced in the absence of Polη (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polζ are required for the efficient bypass of abasic sites in yeast.

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Section: Methodsmentioning

confidence: 99%

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

Kow

Bao²,

Minesinger³

et al. 2005

Nucleic Acids Research

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“…C4-AP undergoes strand scission in free DNA under physiologically relevant buffer conditions considerably more rapidly than AP. 38,55–57…”

Section: Resultsmentioning

confidence: 99%

“…C4-AP, DOB, and AP photochemical precursor phosphoramidites were synthesized as described previously and incorporated into DNA by solid phase oligonucleotide synthesis. 13,37,38 [γ-32 P]dATP and [α-32 P]ATP (cordycepin) were obtained from PerkinElmer. Protein purification was conducted using an AKTA FPLC instrument using columns from GE Healthcare.…”

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confidence: 99%

Expanded Substrate Scope of DNA Polymerase θ and DNA Polymerase β: Lyase Activity on 5′-Overhangs and Clustered Lesions

Laverty

Greenberg

2018

Biochemistry

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DNA polymerase θ (Pol θ) is a multifunctional enzyme with double strand break (DSB) repair, translesion synthesis, and lyase activities. Pol θ lyase activity on ternary substrates containing a 5’-dRP that are produced during base excision repair of abasic sites (AP) is weak compared to DNA polymerase β (Pol β), a polymerase integrally involved in base excision repair (BER). This led us to explore whether Pol θ utilizes its lyase activity to remove 5’-dRP and incise abasic sites from alternative substrates that might be produced during DNA damage and repair. We found that Pol θ exhibited lyase activity on abasic lesions near DSB termini and on clustered lesions. To calibrate the Pol θ activity, Pol β reactivity was examined with the same substrates. Pol β excised 5’-dRP from within a 5’-overhang 80-times faster than did Pol θ. Pol θ and Pol β also incised AP within clustered lesions but showed opposite preferences with respect to the polarity of the lesions. AP lesions in 5’-overhangs were typically excised by Pol β 35–50-times faster than those in a duplex substrate but 15- to 20-fold less than 5’-dRP in a ternary complex. This is the first report of Pol θ exhibiting lyase activity within an unincised strand. These results suggest that bifunctional polymerases may exhibit lyase activity on a greater variety of substrates than previously recognized. A role in DSB repair could potentially be beneficial, while the aberrant activity exhibited on clustered lesions may be deleterious due to their conversion to DSBs.

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“…22,23 In spite of the possible advantages of amine modification, one concern is that the 1,4-dicarbonyl system in 3 could undergo strand cleavage at the β-position of the aldehyde (β-elimination) during the bioconjugation; this would generate ODN fragments that are unsuitable for conjugation. Leumann reported that the strand cleavage of abasic RNA sites and their 2′-O-methylated analogs under alkaline conditions was slower than that observed with abasic DNA.…”

Section: Bioconjugation Of Oligodeoxynucleotides (Odns) With Biomolecmentioning

confidence: 99%

Bioconjugation of Oligodeoxynucleotides Carrying 1,4-Dicarbonyl Groups via Reductive Amination with Lysine Residues

et al. 2015

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We evaluated the efficacy of bioconjugation of oligodeoxynucleotides (ODNs) containing 1,4-dicarbonyl groups, a C4'-oxidized abasic site (OAS), and a newly designed 2'-methoxy analogue, via reductive amination with lysine residues. Dicarbonyls, aldehyde and ketone at C1- and C4-positions of deoxyribose in the ring-opened form of OAS allowed efficient reaction with amines. Kinetic studies indicated that reductive amination of OAS-containing ODNs with a proximal amine on the complementary strand proceeded 10 times faster than the corresponding reaction of an ODN containing an abasic site with C1-aldehyde. Efficient reductive amination between the DNA-binding domain of Escherichia coli DnaA protein and ODNs carrying OAS in the DnaA-binding sequence proceeded at the lysine residue in proximity to the phosphate group at the 5'-position of the OAS, in contrast to unsuccessful conjugation with abasic site ODNs, even though they have similar aldehydes. Theoretical calculation indicated that the C1-aldehyde of OAS was more accessible to the target lysine than that of the abasic site. These results demonstrate the potential utility of cross-linking strategies that use dicarbonyl-containing ODNs for the study of protein-nucleic acid interactions. Conjugation with a lysine-containing peptide that lacked specific affinity for ODN was also successful, further highlighting the advantages of 1,4-dicarbonyls.

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Synthesis and Characterization of Oligonucleotides Containing the C4′‐Oxidized Abasic Site Produced by Bleomycin and Other DNA Damaging Agents

Cited by 18 publications

References 35 publications

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

Expanded Substrate Scope of DNA Polymerase θ and DNA Polymerase β: Lyase Activity on 5′-Overhangs and Clustered Lesions

Bioconjugation of Oligodeoxynucleotides Carrying 1,4-Dicarbonyl Groups via Reductive Amination with Lysine Residues

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