This proposal has been structured to help test the hypothesis that it is the accumulation of damage within DNA that causes some of the phenotypes associated with aging. Specifically, the objective of this application is to determine how DNA repair mechanisms change with aging. Repair of DNA in both the nucleus and the mitochondria will be evaluated in various human cell cultures which are models of aging. The objective of the proposal will be pursued through the following specific aims. i) An examination of the effects of aging on the repair of oxidative damage in DNA. These experiments will evaluate the repair of sugar-phosphate and base lesions caused by reactive oxygen species in both mitochondrial DNA and specific nuclear sequences. 2) A study of the effects of aging on the repair on N- methylpurines. These studies will evaluate the repair of N7-methylguanine and N3-methyladenine in both mitochondrial DNA and specific nuclear sequences. 3) An evaluation of the effects of aging on the repair of 06- methylguanine. Initial studies in this aim will correlate the repair of this mutagenic lesion in the total genome with the levels of 06- methylguanine-DNA-methyltransferase in the cells. Subsequent studies will employ an antibody specific to this lesion and PCR technology to determine the repair of this DNA adduct in mitochondrial DNA and specific transcribed and nontranscribed sequences of nuclear DNA. 4) A determination of the effects of aging on the replication of mitochondrial DNA. These studies will use the incorporation of the heavy base analogue bromodeoxyuridine into DNA during replication. This makes it possible to separate replicated mitochondrial DNA from nonreplicated DNA on cesium chloride gradients so that DNA replication can be quantified. Both the effects of aging alone and in combination with exposure to nitrogen mustard, which produces adducts which are not repaired in mitochondrial DNA, on mitochondrial DNA replication will be studied. When successfully completed,. these studies will provide a more thorough understanding of how the aging process can adversely affect DNA repair mechanisms and cause the accumulation of DNA damage which would lead to impairment of normal cellular functions and/or death.
