DNA-protein crossing-linking by BIS-electrophiles
2008-10
Loading...
View/Download File
Persistent link to this item
Statistics
View StatisticsJournal Title
Journal ISSN
Volume Title
Title
DNA-protein crossing-linking by BIS-electrophiles
Alternative title
Authors
Published Date
2008-10
Publisher
Type
Thesis or Dissertation
Abstract
A key carcinogenic metabolite of the important industrial chemical 1,3-butadiene
(BD), DEB is a bifunctional alkylating agent capable of reacting with both DNA and
proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-
guanine monoadducts, which can then react with a second nucleophilic site to form crosslinked
adducts. A recent report revealed a strong correlation between expression of the
human DNA repair protein AGT in bacteria and the cytotoxic and mutagenic activity of
DEB (J. G. Valadez et al., Chem. Res. Toxicol. 17 (2004) 972-982). As AGT expression
appeared to enhance the toxic effects of this bis-electrophile, the authors proposed that
DEB induces AGT-DNA cross-links. The purpose of our study was to structurally
characterize DEB-induced AGT-DNA conjugates and to identify amino acid residues
within the protein involved in cross-linking. DNA-protein cross-link formation was first
detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were
exposed to DEB in the presence of both wild-type AGT or a C145A AGT mutant.
Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT
that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG
monoepoxide) revealed the ability of the protein to form either one or two butanediol- dG
cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+-
MS/MS sequencing of tryptic peptides obtained from dG monoepoxide-treated AGT
indicated that the two cross linking sites were located at the alkyl acceptor site, Cys145
39
and a neighboring active site residue, Cys150. The same two active site cysteines became
cross-linked to DNA following DEB treatment. Modification of Cys145 was further
confirmed by HPLC-ESI+-MS/MS analysis of dG monoepoxide-treated synthetic peptide
GNPVPILIPCHR which represents the active site tryptic fragment of AGT (C = Cys145 ).
Replacement of the catalytic cysteine residue with alanine (C145A AGT) abolished
DEB-induced cross-linking at this site, while the formation of conjugates via neighboring
Cys150 was retained. The exact chemical structure of the cross-linked lesion was
established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+-MS/MS
analysis of the amino acids resulting from total digestion of modified AGT analyzed in
parallel with an authentic standard. Based upon these results, the formation of AGT-DNA
cross-links is a likely mechanism to explain the enhanced cytotoxicity of DEB in cells
expressing this important repair protein.
Description
University of Minnesota Ph.D. dissertation. October 2008. Major: Medicinal chemistry. Advisor: Dr. Natalia Tretyakova. 1 computer file (PDF); xiv, 239 pages, includes col. illustration.
Related to
Replaces
License
Collections
Series/Report Number
Funding information
Isbn identifier
Doi identifier
Previously Published Citation
Other identifiers
Suggested citation
Loeber, Rachel Lea. (2008). DNA-protein crossing-linking by BIS-electrophiles. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/46893.
Content distributed via the University Digital Conservancy may be subject to additional license and use restrictions applied by the depositor. By using these files, users agree to the Terms of Use. Materials in the UDC may contain content that is disturbing and/or harmful. For more information, please see our statement on harmful content in digital repositories.