Publication Date

2015

Document Type

Honors Thesis

Department

Biochemistry

Keywords

DNA-Structure, Nuclear magnetic resonance spectroscopy, DNA repair, Precancerous conditions, DNA, Sp lesion, Base-pair opening, 2D NMR, NOESY, Nuclear Overhauser effect spectroscopy

Abstract

Exposure to transition metals can damage DNA by oxidizing its nucleobases. Guanine, which has the lowest reduction potential of the DNA bases, is particularly vulnerable to the formation of lesions. The spiroiminodihydantoin (Sp) lesion, a hyperoxidized form of guanine, is especially mutagenic, and it can lead to cancer when left unrepaired (Khutsishvili et al., 2013). My research investigates the specific structural effects of the Sp lesion on an 11-mer DNA duplex using 1H Nuclear Magnetic Resonance (NMR) spectroscopy experiments. Two-dimensional Nuclear Overhauser Effect Spectroscopy (2D-NOESY) experiments show the through-space interactions between protons that are five angstroms or fewer apart. These cross-peaks, between the interacting protons, form predictable patterns in undamaged DNA duplexes; deviations from this pattern indicate structural damage (e.g. puckering, twisting) (Tang et al., 2012). Additional 1H NMR experiments called base-pair opening experiments have been performed in order to learn more about the Sp-lesion containing duplex. These experiments use integrations of one-dimensional (1D) 1H NMR spectra to measure the solvent-exchange rates of the imino protons in the duplexes. These rates are lower in stable duplexes, because the conservation of Watson-Crick base-pairing minimizes the imino protons' exposure to the solvent (Crenshaw et al., 2011). The goals of my honors thesis project are to use 2D NMR spectroscopy to elucidate the structural damage caused by the oxidation of a single nucleobase (G6) in an 11-mer DNA duplex, and to use NMR base-pair opening experiments to understand the kinetic difference between the opening and closing of the nitrogenous DNA bases in Spcontaining and control duplexes.

Language

English

Comments

81 pages : illustrations (some color). Honors project-Smith College, 2015. Includes bibliographical references (pages 78-81)

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