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Title page for ETD etd-02102015-155818

Type of Document Dissertation
Author Stavros, Kallie Marie
Author's Email Address kallie.m.stavros@vanderbilt.edu
URN etd-02102015-155818
Title Conformation of the Aryl Amine DNA Adduct 2-Amino-3-methylimidazole-[4,5-f]quinoline N2-deoxyguanine in the NarI Endonuclease Recognition Sequence as Determined by NMR Spectroscopy
Degree PhD
Department Chemical and Physical Biology
Advisory Committee
Advisor Name Title
Brandt Eichman Committee Member
Carmelo Rizzo Committee Member
Charles Sanders Committee Member
Frederick Guengerich Committee Member
Michael Stone Committee Member
  • structural biology
  • NMR
  • DNA adducts
  • heterocyclic amines
Date of Defense 2014-12-18
Availability unrestricted
2-Amino-3-methylimidazo-[4,5-f]quinoline, IQ, has been identified as one of the most genotoxic materials according to Ames assays and animal studies. IQ is during high temperature cooking of meats and is classified as probably human carcinogen by the IARC. The N2-dG adduct has been shown to have a sequence dependence when replicated by Polη when located in a GCGC repeat sequence. This project investigates differences between the adduct located at the G1 position of the 12mer containing the NarI recognition sequence (G1G2CG3CC) as well as the mutagenic hotspot G3 position using high-field solution NMR. Overall, NMR data supports both adducts adopt a base-displaced intercalated in the duplex, where the IQ spans across the duplex and stacks with the flanking base pairs. The modified bases are displaced into the major groove, while the base opposite the modification is completely flipped into the major groove. Both modified bases maintain the anti conformation. Both adducts do not change the thermal stability of the duplex. The IQ moiety at G3 position, however, is angled by 15° relative to the X7 base, whereas the IQ at G1 remains planar to X4. Further studies are needed to determine if and how this might be significant during replication, but if the orientation is maintained in the polymerase active site, it could play a role in the context determining mutagenicity. This project also differentiates the N2-dG adduct from the major C8-dG adduct, where the N2-dG-IQ has been observed to be more persistent in tissues and provides insight to how it might evade repair by the nucleotide excision repair pathway. This likely relates to the ability of IQ to participate in base-stacking in the duplex when attached at the N2 position of guanine as well as how the modified base remains in the anti conformation. The C8-dG-IQ, however, changes to the syn conformation. The orientation of the C8-dG-IQ results in destabilizing perturbations to the helix that are reflected in the thermal stabilities. These differences could likely contribute to the differing repair response for these IQ adducts.
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