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The analysis of DNA oxidation and study of DNA-Protein cross-links by PAGE and LC-Mass Spectrometry

Nemera, Dessalegn B
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1407406995

Abstract Details

2014, MS, University of Cincinnati, Arts and Sciences: Chemistry.
Various endogenous and exogenous agent including free radicals from normal cellular metabolisms, UV light, ionizing radiation and metal complexes attack biomolecules, resulting in DNA and protein oxidation. Amongst these biomolecules, DNA oxidation is genotoxic and a very important area of research into understanding the mechanisms behind what causes ageing, cell death, cancer and other diseases. Damage caused to DNA can result in mutations, strand breaks and cross-linking between DNA and protein. To understand what causes these defects, how they occur and what damage they actually do to DNA is therefore very important. Of those various types of oxidized DNA lesions, my research mainly focuses on DNA-protein cross-link also called DPC, bulky DNA lesions that are expected to interfere with normal DNA-protein interaction. During the formation of this lesion, proteins are covalently trapped on DNA when cells are exposed to DNA-damaging agents. Oxidative DNA-protein cross-links are readily formed in biologically relevant oxidation systems and have received less attention than other types of DNA damage. Cross-link formation was examined using four different oxidation systems that generate singlet oxygen, superoxide, and metal-based Fenton reactions. Our lab detected and characterized DPCs using HPLC-mass spectrometry and Polyacrylamide gel electrophoresis (PAGE) by identifying the site of cross-linking and the structures of adducts involved. Because cross-links are inherently complex, we initially identified adducts formed from small molecules consisting of amino acids and guanine to larger systems involving ribonuclease A and a 27-nucleotide DNA. Our findings indicate that oxidative cross-links predominantly dependent on the number of guanines on the 27-nucleotide DNA sequence. The results additionally suggest that the guanine content on the formation of oxidative lesions is a strong predictor of overall oxidative DNA damage. There is also a significant level of oxidative cross-linking that occurs between guanine and nucleophilic amino acids of a protein. This result implies that cross-links may be present in high levels in the cells since the propensity to oxidatively cross-link is high and much of the genomic DNA is coated with proteins. The two methods described on this thesis allow for the detection and characterization of DPC under various environmental and experimental conditions.
Edward Merino, Ph.D. (Committee Chair)
Patrick Limbach, Ph.D. (Committee Member)
Peng Zhang, Ph.D. (Committee Member)
102 p.
Biochemistry
DNA oxidation; DNA-protein cross-links; PAGE; LC-mass spectrometry

Recommended Citations

Citations

  • Nemera, D. B. (2014). The analysis of DNA oxidation and study of DNA-Protein cross-links by PAGE and LC-Mass Spectrometry [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1407406995

    APA Style (7th edition)

  • Nemera, Dessalegn. The analysis of DNA oxidation and study of DNA-Protein cross-links by PAGE and LC-Mass Spectrometry. 2014. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1407406995.

    MLA Style (8th edition)

  • Nemera, Dessalegn. "The analysis of DNA oxidation and study of DNA-Protein cross-links by PAGE and LC-Mass Spectrometry." Master's thesis, University of Cincinnati, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1407406995

    Chicago Manual of Style (17th edition)

ucin1407406995
801
© 2014, some rights reserved.Creative Commons License The analysis of DNA oxidation and study of DNA-Protein cross-links by PAGE and LC-Mass Spectrometry by Dessalegn B Nemera is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Cincinnati and OhioLINK.