| 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 form 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 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 (NER) 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 mechanism s of repair of oxidative DNA base damages. Using in vitro assays we are able to examine the repair of different types of lesions and to measure each different step of the pathway. Furthermore, we can measure damage processing in the nuclear and mitochondrial compartments separately. In vitro assays with nuclear extracts from cells and substrates containing oxidized base damages show that there are two forms of BER, the short and long patch, distinguishable by the length of the newly incorporated bases in DNA. We have shown that DNA polymerase beta is involved in both of these processes, and that it plays a critical role, possibly in the interaction with the flap-endonuclease FEN-1. Our results suggest that a major base excision repair complex exists, which includes AP endonuclease, proliferating cell nuclear antigen (PCNA), polymerase (s), glycosylase(s) and possible other proteins. These protein interactions are physical and functional and together support the "passing of baton" model, in which base excision repair takes place in different steps supported by individual protein interactions that are components of a repair complex, possibly situated at the DNA lesion. |
| Cockayne syndrome (CS) is a rare human genetic disease, characterized by a premature aging phenotype. 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 (OGG1) protein, which is the major enzyme for the repair of the highly prevalent oxidized base 8-hydroxyguanosine (8-OHdG). 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. |
| The 8-OHdG base lesion is of special interest since it is formed at high levels after oxidative stress and it causes mutations, if left unrepaired. We have studied the mechanism of repair of this lesion and find that it is repaired mainly via the BER pathway. In addition, 8OHdG is repaired in a mode that is not coupled to transcription both in the nucleus and in mitochondria. We have also investigated the role of the oxoguanine DNA glycosylase 1 (OGG1) in mtDNA repair in mice that are defective in this enzyme. We found that liver mitochondria from the OGG1 knockout mice have no detectable 8OHdG incision activity, demonstrating that the mitochondrial activity is encoded by the same gene as the nuclear enzyme. Mitochondrial DNA from the knockout mice accumulates 9 times more 8OHdG than wt animals. In contrast, nuclear DNA from the same animals has only two times more 8-OHdG modifications than controls. These results suggest that OGG1 plays a crucial role in the repair of oxidative damage in mitochondria and is probably the only 8OHdG glycosylase in these organelles. Using a similar approach, mice deficient in the DNA glycosylase NTH1, we investigated the repair of oxidized pyrimidines in mitochondria. We have shown that NTH1 is the major enzyme for the repair of oxidative base damage to pyrimidines and that OGG1 does not play a significant role in this pathway.
We have previously reported that there is an increase with age in OGG1 activity, from 6 to 23 months of age, in liver mitochondrial extracts from rats and mice. In contrast, two other mitochondrial enzymes of DNA metabolism which are not specifically involved in the repair of oxidative damage, uracil DNA glycosylase (mtUDG) and AP endonuclease, had no change in activity with aging. While mitochondrial DNA repair increased with age, the nuclear DNA repair slightly decreased in the same animals. Since caloric restriction (CR) is the only known intervention to slow the progression of aging, we investigated BER activities in caloric restrict old mice. We found that BER is regulated by caloric restriction differently in nucleus and mitochondria. While nuclear BER was significant increased in CR mice, mitochondrial BER was only marginally affect, in a lesion specific manner. These results may reflect the reduced levels of oxidative stress in mitochondria from CR animals. We are studying DNA repair of oxidative DNA lesions in caloric restricted mice to determine whether DNA repair is affected by such dietary changes. We are also subjecting knockout mice, defective in specific DNA repair genes, to caloric restriction. This is another approach to determine whether DNA repair plays a role in this process. |
| The p53 protein has recently been associated with BER in the nucleus. We investigated whether p53 participates in BER in mitochondria and found that mitochondrial extracts from p53 null mouse liver have normal levels of DNA glycosylase activity but a slightly reduced DNA repair synthesis incorporation. DNA polymerase gamma activity was lower in absence of p53, but addition of recombinant p53 complemented this defect. Our results suggest that p53 plays a role in mitochondrial BER at the incorporation step. Thus, p53 translocation to mitochondria may modulate BER up-regulation in response to stress. |
| To investigate the tissue specificity of BER we measured DNA glycosylase actvities in nuclear and mitochondrial extracts from mouse testis, liver, kidney, muscle, brain and heart. Testis had the highest BER levels in both nucleus and mitochondria, suggesting that BER plays a critical role in maintaining genetic integrity. Our results show that BER levels vary greatly among the different organs. We are now investigating BER activities in different brain regions of normal mice, and of mice models of various age-associated neurodegenerative diseases. These studies may provide valuable insight into the mechanisms that lead to neuronal loss with age. |
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