Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions.
We report on the elucidation of DNA-protein cross-links formed in isolated mammalian chromatin upon treatment with H2O2 in the presence of iron or copper ions. Analysis of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization showed the presence of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine (thymine-tyrosine cross-link) on the basis of the gas chromatographic and mass spectrometric characteristics of the trimethylsilylated authentic compound. Other DNA-protein cross-links involving thymine and the aliphatic amino acids and cytosine and tyrosine, which were known to occur in nucleohistone gamma-irradiated under anoxic conditions, were not observed. This was due to inhibition by oxygen as clearly shown by experiments that were carried out using ionizing radiation under both oxic and anoxic conditions instead of using H2O2 and metal ions. However, oxygen did not inhibit formation of the thymine-tyrosine cross-link in gamma-irradiated chromatin or in chromatin treated with H2O2 and metal ions. The yield of the thymine-tyrosine cross-link was higher upon treatment with H2O2/chelated Fe3+ ions than with H2O2/unchelated Fe3+ ions. By contrast, H2O2/unchelated Cu2+ ions produced a higher yield than H2O2/chelated Cu2+ ions. Almost complete inhibition of cross-link formation was provided by the hydroxyl radical scavengers mannitol and dimethyl sulfoxide when H2O2/chelated metal ions were used. On the other hand, scavengers only partially inhibited formation of cross-links when H2O2/unchelated metal ions were used, possibly indicating the site-specific nature of cross-linking. Superoxide dismutase afforded partial inhibition only when chelated ions were used.(ABSTRACT TRUNCATED AT 250 WORDS)
Nackerdien
Z
Z
Chemical Science & Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
Rao
G
G
Cacciuttolo
M A
MA
Gajewski
E
E
Dizdaroglu
M
M
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects*; D003300:Copper / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006657:Histones / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007501:Iron / Q000494:pharmacology*; D051379:Mice; D011485:Protein Binding
D002843:Chromatin; D003432:Cross-Linking Reagents; D006657:Histones; D003300:Copper; D004247:DNA; D006861:Hydrogen Peroxide; D007501:Iron