Biological Consequences Of Complex Dna Lesions Induced By Bis-Electrophiles
2014-08
Loading...
View/Download File
Persistent link to this item
Statistics
View StatisticsJournal Title
Journal ISSN
Volume Title
Title
Biological Consequences Of Complex Dna Lesions Induced By Bis-Electrophiles
Authors
Published Date
2014-08
Publisher
Type
Thesis or Dissertation
Abstract
Genomic DNA is constantly modified by bis–electrophiles which induce a wide array of DNA adducts. DNA adducts can threaten cell viability and genomic integrity by interfering with DNA replication and transcription. Unless repaired, they can be misread by DNA polymerases, leading to heritable mutations and cancer. Amongst known human carcinogens is 1,3–butadiene (BD), an important industrial chemical and environmental pollutant. BD is metabolized to reactive epoxides that induce a range of DNA adducts. In the first part of this thesis, we investigated the ability of human cells to repair three potentially mutagenic BD–adenine lesions: N 6–(2–hydroxy–3–buten–1–yl)–2 ’–deoxyadenosine, 1,N 6–(2–hydroxy–3–hydroxymethylpropan–1,3–diyl)–2 ’–deoxyadenosine and N 6–,N 6–(2,3–dihydroxybutan–1,4–diyl)–2 ’–deoxyadenosine. Repair assays using site– and stereo–specifically adducted oligodeoxynucleotides revealed that all three BD–dA adducts were recognized by base excision repair (BER) pathway. Repair inhibition by a BER inhibitor, and 5’ excision products characterized by HPLC–ESI––MS/MS analysis confirmed the involvement of BER.
Exposure to bis–electrophiles or reactive oxygen species can irreversibly trap cellular proteins on DNA, forming super–bulky lesions known as DNA–protein cross–links (DPCs). The complexity and diversity of DPCs have prevented researchers from investigating their biological effects. In the second part of this thesis, we have developed bioconjugation methods to generate site–specific DPCs and examined their effects on DNA replication. Hydrolytically stable 7–deazaguanine adducted DPCs, which are structurally analogous to cellular DPCs induced by antitumor nitrogen mustards, were prepared by post–synthetic reductive amination. Further, Huisgen cycloaddition was used to generate DPCs involving the C5–thymidine of DNA. Replication bypass using human translesion synthesis polymerases η and κ revealed that large polypeptides cross–linked to either 7–deazaguanine or C5–thymidine completely blocked replication, while the corresponding decapeptide cross–links were bypassed, suggesting that bypass of DPCs in cells is mediated by proteolytic degradation of the cross–linked proteins. Steady–state kinetic studies provided evidence for the highly error–prone bypass of C5–thymidine peptide cross–links and error–free bypass of N7–guanine cross–linked peptides. Further, HPLC–ESI––MS/MS characterization of replication products of C5–thymidine peptide cross–links revealed large numbers of deletion and substitution mutations. Taken together, our data suggest that the efficiency and the fidelity of DNA replication past DPCs are dependent on the lesion size, the cross–linking site and DNA polymerase identity.
Description
University of Minnesota Ph.D. dissertation. August 2014. Major: Chemistry. Advisors: Natalia Tretyakova, Mark Distefano. 1 computer file (PDF); xxxiii, 337 pages.
Related to
Replaces
License
Collections
Series/Report Number
Funding information
Isbn identifier
Doi identifier
Previously Published Citation
Suggested citation
Wickramaratne, Susith. (2014). Biological Consequences Of Complex Dna Lesions Induced By Bis-Electrophiles. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/174906.
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.