Homogeneous preparations of 3'-phosphoglycolate-terminated oligodeoxynucleotides from bleomycin-treated DNA as verified by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.
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.
Chen
S
S
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA.
Hannis
J C
JC
Flora
J W
JW
Muddiman
D C
DC
Charles
K
K
Yu
Y
Y
Povirk
L F
LF
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D006016:Glycolates / Q000378:metabolism*; D013058:Mass Spectrometry; D009841:Oligonucleotides / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
D000964:Antimetabolites, Antineoplastic; D006016:Glycolates; D009841:Oligonucleotides; D001761:Bleomycin; D004247:DNA; D000469:Alkaline Phosphatase; C013193:phosphoglycolate