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Title page for ETD etd-03252014-080514


Type of Document Dissertation
Author Szulik, Marta Wieslawa
URN etd-03252014-080514
Title Oxidation of Cytosine in DNA
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Michael P. Stone Committee Chair
Carmelo Rizzo Committee Member
Martin Egli Committee Member
Terry P. Lybrand Committee Member
Keywords
  • DNA; cytosine oxidation; DNA methylation; DNA demethylation; BER; NMR; X-ray crystallography; base p
Date of Defense 2014-02-27
Availability unrestricted
Abstract
The search for active demethylation processes lead to discovery of 5-hydroxymethylcytosine (5hmC) adduct in the mammalian DNA. This lesion is formed post-replicatively by oxidation of 5methylcytosine (5mC) in the presence of TET deoxygenases. Futher oxidation products of TET oxidations, 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) are substrates for base excision repair and removed by Thymine DNA Glycosylase (TDG). However up to date it is still unknown which enzyme is responsible for 5hmC removal from DNA. In this work, studies of the modified Dickerson-Drew Dodecamer 5'-CGC GAA TTX GCG-3' (DDD) are presented, where X is 5mC, 5hmC, 5fC or 5caC. Structural studies by X-ray crystallography revealed that the base pairing and base stacking is maintained in analyzed DNA duplexes. The NMR as a function of temperature describes the effect induced by modified bases on the DNA stability. High-resolution proton exchange NMR spectroscopy has been used to characterize kinetics of base-pair opening in nucleic acid duplexes. The rates of exchange of imino protons with solvent protons were measured by magnetization transfer from water for each DNA duplex, as a function of the concentration of exchange catalyst. It was proposed that TDG uses an extrahelical base recognition mechanism, which is facilitated by destabilized base pair possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of DNA containing 5mC, 5hmC 5fC and 5caC are crucial to understand the role of repair enzyme in damage recognition. In work presented herein the similarities and differences among all adducts are discussed, as well as their potential as a substrates for base excision repair mechanisms.
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