| Oxidative DNA Damage and Its Processing: Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can cause mutations and genomic instability. Many different DNA base changes have been seen following some forms of oxidative stress. These lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is in mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms responsible for the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair pathways, and there is currently a great deal of interest in understanding how these pathways operate and interact. We have used a number of different approaches to explore the mechanisms of repair of oxidative DNA base damage. Using in vitro assays, we are able to examine the repair of different types of lesions and to separately measure each step of the BER pathway. Our results show that BER levels vary greatly among the different mouse organs. Furthermore, we can measure damage processing in the nuclear and mitochondrial compartments separately and have made major contributions towards defining the types of DNA lesions repaired in mitochondria. |
| Cockayne syndrome (CS) is a rare human genetic disease, characterized by premature aging phenotypes. We have demonstrated that cells from CS patients are deficient in the repair of oxidative lesions, and that they accumulate such lesions in their DNA after oxidative stress induced by gamma irradiation. We further showed that CSB, one of the proteins mutated in CS patients, interacts with the oxoguanine DNA glycosylase protein, which is the major enzyme for the repair of the highly prevalent oxidized base 8-hydroxyguanosine. In correlation to this observation, repair of oxidative damage is also defective in mitochondrial DNA from CS cells, and this may be the major underlying cause of the disease. Mitochondrial bioenergetics are also disrupted in CSB cells. Using a variety of techniques, we have measured elevated mitochondrial oxygen consumption in CSB-deficient cells and determined that CSB mitochondria preferentially burn fat. This finding may have important implications for the cachectic dwarfism observed in CS patients and we are testing the hypothesis that a high fat diet may be beneficial for these individuals. In other ongoing studies, we are exploring the relationship between DNA repair and mitochondrial bioenergetics. We seek to test the hypothesis that dysfunctional DNA repair (either nuclear or mitochondrial) may lead to altered cellular bioenergetics requirements, mitochondrial dysfunction and ultimately to premature aging. |
