Oxidative Dna Damage And Its Processing

Aging

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Abstract

Summary of work: 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 resyn-thesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as in- stigators 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 involved in 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 cur-rently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to meas-ure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. We have established in vitro assays using nuclear extracts from cells to measure the process of base excision repair of oxidative DNA damage. The experiments show that there are two forms of Base excision repair distinguishable by the length of the newly synthesized bases in DNA. We have shown that DNA polymerase beta is in volved in both of these processes, and that it plays a critical role, possibly in interaction with FLAP endonuclease FEN-1. We have disclosed a number of other protein interaction between proteins involved in the Base excision repair pathway. We have reconstituted this process in vitro and are exploring both physical and functional protein interactions. This suggests that a major base excision repair complex exists which includes AP endonuclease, proliferating cell nuclear antigen (PCNA), polymerase (s), glycosylase(s) and others. These protein interactions are physical and functional and together support the "passing of buton" 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. We are further examining these inetractionsIn a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.