The long range objective of this project is to develop a more thorough understanding of the cellular mechanisms which are involved in the processing of environmentally induced DNA damage. These repair mechanisms are crucial for the maintenance of genomic stability. Until recently, DNA repair experiments were limited to investigations concerning the entire genome. With the utilization of molecular biological techniques, it is now possible to evaluate repair within specific DNA sequences. Such analysis of sequence-specific repair has revealed a heterogeneity in the rate of removal of DNA damage across the mammalian genome. To date this work, like most DNA repair experiments, has been conducted in proliferating cells grown in vitro. However, the vast majority of mammalian cells in situ are differentiated and exhibit a greatly reduced replicative capability. It has been suggested that the repair capacity of differentiated cells may be unlike that of rapidly proliferating cells. In fact, a reduced DNA repair efficiency has been associated with differentiation. This would consequently increase the vulnerability of these cells to environmental insults. Because of the vital role that differentiated cells play in normal human function, it is important to understand the influences of differentiation on DNA repair mechanisms. Therefore, this proposal seeks to investigate the effects of differentiation on repair of alkylation damage. The goal of this proposal will be pursued through the following specific aims: 1) A determination of the effects of differentiation on the formation and removal of alkali- labile sites across the entire genome. 2) A determination of the effects of differentiation on the formation and removal of O6-methylguanine across the entire genome. 3) A determination of the effects of differentiation on the formation and removal of alkali-labile sites within transcribed and nontranscribed nuclear DNA sequences. 4) A determination of the effects of differentiation on the formation and repair of alkylation damage within mitochondrial DNA These studies will be performed using primary cultures of oligodendrocytes which in culture proceed through a very well-defined, time-dependent cascade of differentiation events. When successfully completed, these studies will broaden our understanding of the factors which influence repair of DNA damage induced by environmental agents.
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