| A consequence of aerobic respiration is a highly oxidizing environment inside of cells. The byproducts of the cellular energy production include reactive oxygen species which damage cellular components. As a result, all organisms contain elaborate antioxidant systems to counter the deleterious effects of oxidation. DNA is susceptible to oxidation, producing a variety of cytotoxic and/or mutagenic lesions, including 8-oxoguanine (oxoG). oxoG is an endogenous mutagen based on its ability to mis-pair with adenosine during DNA replication, giving rise to transversion mutations. Oxidative damage has been implicated in human ailments such as cancer, aging and neurological disease. This thesis describes efforts to understand the mechanism of oxoG repair in eukaryotes.;The first part of this thesis describes the study of oxidative DNA damage repair in the yeast Sacchromyces cerevisiae, a eukaryotic model organism. DNA glycosylases are responsible for the removal of a majority oxidative DNA damage. Yeast have a glycosylase specific for oxoG:C (yOgg1), but unusually contain a second activity that cleaves oxoG opposite any of the four bases (yOgg2). This activity was biochemically isolated and identified from yeast using a mechanism-based cloning strategy. Based on the characteristics of yOgg1 and yOgg2, a novel system of oxoG repair in yeast was proposed. Yeast genetics and mutagenesis experiments support this hypothesis.;Inhibitors of DNA repair glycosylases have found wide use in elucidating enzyme mechanism and in isolating novel proteins using affinity techniques. The novel abasic site analog the ring-opened pyrrolidine was designed based on a transition-state model and the structures of known inhibitors. This general inhibitor for DNA glycosylases was synthesized and evaluated based on binding to hOGG1.;The human 8-oxoguanine glycosylase (hOGG1) is a homolog of the yeast yOgg1 enzyme, with specificity for oxoG paired opposite C. Aberrant (hOGG1) function in humans has been implicated in the development of cancers and in aging. In order to gain detailed information on (hOGG1) function, the structure of an enzyme/DNA complex was solved to 2.1Å using MAD phasing techniques. |
