Homogeneous Preparations of 3′-Phosphoglycolate-Terminated Oligodeoxynucleotides from Bleomycin-Treated DNA as Verified by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
✍ Scribed by Shuang Chen; James C. Hannis; Jason W. Flora; David C. Muddiman; Kwabena Charles; Yin Yu; Lawrence F. Povirk
- Publisher
- Elsevier Science
- Year
- 2001
- Tongue
- English
- Weight
- 143 KB
- Volume
- 289
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Single- and double-strand breaks bearing 3'-phosphoglycolate termini are among the most frequent lesions formed in DNA by ionizing radiation and other oxidative mutagens. In order to obtain homogeneous preparations of defined 3'-phosphoglycolate substrates for repair studies, 5'-(32)P-end-labeled partial duplex DNAs were treated with bleomycin, and individual cleavage products were isolated from polyacrylamide gels. The fragments were then treated with alkaline phosphatase and further purified by reverse-phase HPLC. Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry of the purified oligomers produced molecular ions of the expected masses, with no detectable contaminants. Gas-phase sequencing by tandem mass spectrometry of these single species yielded the expected sequence ions and confirmed the presence of phosphoglycolate on the 3'-terminal fragments only. The fragments could be relabeled with polynucleotide kinase to yield highly purified, high-specific-activity substrates for repair studies.