Cellular responses to oxidative stress are a critical line of defense against reactive oxygen species that alter the structure of DNA bases leading to mutagenesis. Errors in the DNA sequence and impaired repair or signaling of DNA damage underlies numerous disorders including cancer, Alzheimer's disease and metabolic disorder. This program has uncovered the chemistry and biochemistry of hyperoxidized guanines including spiroiminodihydantoin, guanidinohydantoin and 2-iminohydantoin. These heterocyclic lesions form in G-rich sequences particularly those capable of folding into a G-quadruplex (G4). The processing of hydantoin lesions in double-stranded DNA vs. G4-DNA appears to be quite different with various members of the NEIL family participating. We hypothesize that the hydantoins in G-quadruplex structures such as promoter sequences function as a sensing mechanism for up-regulation of repair enzymes involved in oxidative stress. This project aims to examine the structures and stabilities of G-quadruplex-forming DNA sequences as a function of oxidative stress, and to examine their binding interactions and glycosylase kinetics with base excision repair proteins. The approach involves synthesis of oligodeoxynucleotides containing site-specifically incorporated G oxidation (8-oxoguanosine or the hydantoins) and to examine the thermal stability, circular dichroism spectra, and the ability to bind to NEIL1-3 or NTHL1. In addition, the ability of damage- containing G4-DNA to induce or inhibit transcription will be studied in a reporter plasmid assay. Innovative concepts and methods include the study of numerous predicted G4 sequences that may play regulatory roles in BER enzyme expression and the suggestion that oxidized bases in DNA could serve 'epigenetic-like' roles in transcription. Novel methods of studying G- quadruplex structure include the use of an ion channel protein, alpha-hemolysin, as a single- molecule reporter of structure for assaying a collection of equilibrating species. The significance of this work lies in uncovering the molecular basis for diseases related to oxidative stress, with a particular focus on the chemistry of the oxidatively damaged DNA and how it triggers structural reorganization in DNA to choreograph downstream events leading to mutation or repair.
Free radicals and oxidative stress are contributors to cancer and other age-related disorders. This work will examine the chemistry of oxidation of the DNA base guanine in guanine-rich sequences such as those found at the ends of chromosomes and in gene promoter regions. It will test the hypothesis that changes in DNA folding induced by oxidative stress play a role in regulating protein expression.
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