Reactive oxygen species (ROS) in the cell lead to DNA damage including base lesions that are mutagenic if unrepaired. The molecular events leading to mutagenesis now provide a direct link between oxidative DNA damage and cancer. An understanding of the molecular basis of carcinogenesis is the foundation of prevention and treatment. The unusual heterocycles 5-guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), whose structures were recently assigned in this laboratory, are major products of guanosine and 8-oxo-7,8-dihydroguanosine (OG) oxidation by a wide variety of (ROS) including singlet oxygen and peroxyl radicals. In the past funding period, these laboratories have investigated the chemical mechanism of formation of Gh and Sp, developed methods of generating highly pure synthetic oligodeoxynucleotides containing these lesions, studied misinsertion of nucleotides opposite the lesions as well as repair by base excision repair (BER) enzymes, and observed a 99 percent mutation rate of both lesions in an in vivo assay. The recent detection of Sp in repair-deficient bacterial cells increases the relevancy of these lesions to oxidative damage and disease.
The aims of the next period seek to understand the molecular basis for the extremely high mutation rates and the unusual G to C mutations observed with Gh and Sp. Other oxidized purines including OG will be studied for comparison when appropriate. Specifically, this work will (1) determine the structures of Gh and Sp in duplex DNA using NMR and x-ray crystallographic techniques, (2) examine misinsertion of nucleotides opposite the lesions with Y-family (lesion bypass) DNA polymerases and investigate sequence-dependent frameshift mutagenesis with eukaryotic polymerases, (3) study the molecular mechanisms of repair of Gh and Sp lesions using BER glycosylases, and (4) study in vivo mutagenesis using site-specifically incorporated lesions in DNA plasmids. All of the specific aims require continual refinement of our synthetic procedures for generation of nucleoside standards, nucleotide triphosphates and oligonucleotide substrates as well as mass spectrometric characterization of the lesions. Collaborators will assist with NMR and x-ray crystallography, and in vivo mutagenesis studies in E. coli. This laboratory will continue to aid collaborators who are developing analytical methods to determine the cellular levels of the Sp and Gh lesions.
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