Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i...

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Bibliographic Details
Published in:Nucleic acids research 2000-03, Vol.28 (6), p.E16-16
Main Authors: Jaruga, P, Speina, E, Gackowski, D, Tudek, B, Olinski, R
Format: Article
Language:English
Subjects:
8-oxo-guanine
Animals
Cattle
Deoxyribonuclease (Pyrimidine Dimer)
DNA - metabolism
DNA Damage
DNA Repair
DNA-Formamidopyrimidine Glycosylase
Endo III endonuclease
Endodeoxyribonucleases - metabolism
Escherichia coli Proteins
FapyA protein
FapyG protein
Fpg endonuclease
Free Radicals
Gas Chromatography-Mass Spectrometry
Humans
N-Glycosyl Hydrolases - metabolism
NAR Methods Online
Oxidation-Reduction
Rats
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Summary:GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.
ISSN:1362-4962
0305-1048
1362-4962
DOI:10.1093/nar/28.6.e16