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) 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.
Jaruga
P
P
Department of Clinical Biochemistry, The L. Rydygier Medical University, ul. Karlowicza 24, 85-092 Bydgoszcz, Poland.
Speina
E
E
Gackowski
D
D
Tudek
B
B
Olinski
R
R
D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D005609:Free Radicals; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats
D029968:Escherichia coli Proteins; D005609:Free Radicals; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase