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Document Type:Latin Dissertation
Language of Document:English
Record Number:52449
Doc. No:TL22403
Call number:‭3330512‬
Main Entry:Rachel Lea Loeber
Title & Author:DNA-protein cross-linking by bis-electrophilesRachel Lea Loeber
College:University of Minnesota
Date:2008
Degree:Ph.D.
student score:2008
Page No:255
Abstract:The genotoxic effects of bis -electrophiles are generally attributed to their ability to cross-link biomolecules. While DNA-DNA cross-linking mediated by 1,2,3,4-diepoxybutane (DEB) and antitumor nitrogen mustards is well characterized and thought to be largely responsible for their cytotoxic effects, the association between DNA-protein cross-linking and genotoxicity is less clear. Acting as bulky, helix-distorting lesions, DNA-protein cross-links block binding and progression of protein complexes, potentially interfering with crucial cellular processes. In the current work, the ability of DEB and nitrogen mustards to induce DNA-protein cross-links was examined in vitro. DEB- and nitrogen mustard-induced cross-linking was first established between double-stranded oligodeoxynucleotides and the human DNA repair protein O6 -alkylguanine DNA alkyltransferase (AGT). Cross-link formation was confirmed by SDS-PAGE, and tandem mass spectrometry was employed to elucidate cross-link structures. Next, the ability of DEB and nitrogen mustards to cross-link AGT to DNA in the presence of other cellular proteins was examined via Western blot analysis and tandem mass spectrometry. The effect of AGT expression in DEB- and nitrogen mustard-induced cytotoxicity and mutagenicity was then investigated in Chinese hamster ovary cells expressing the human protein. Lastly, additional proteins that had become cross-linked to DNA in the presence of the nitrogen mustard mechlorethamine were identified using a combination of Western blot analysis and shot-gun proteomics. As we have demonstrated that DNA-protein cross-links form upon exposure to DEB and nitrogen mustards, cross-links of this type are likely to contribute to the cytotoxicity of bis -electrophiles in mammalian cells.
Subject:Health and environmental sciences; Pure sciences; Bis-electrophiles; Cross-linking; Mass spectrometry; Nitrogen mustards; Toxicology; Analytical chemistry; Biochemistry; 0486:Analytical chemistry; 0383:Toxicology; 0487:Biochemistry
Added Entry:N. Tretyakova
Added Entry:University of Minnesota