Oxidative Damage to DNA Repair Pathways

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DESCRIPTION (provided by applicant): Redox regulation is a term describing modification of function by a change in the state of oxidation vs. reduction of critical control molecules, usually proteins. When cells are subjected to reactive oxygen species in a process described as 'oxidative stress', pathways susceptible to redox regulation may be altered. Defining the mechanism of such alteration is often difficult because oxidative stress can additionally cause damage to multiple cellular targets, including membrane, organelles and chromatin, often leading to mitotic or apoptotic death. Our proposed experiments will allow a separation of damage to the redox-regulated proteins from that of damage to targets of cytotoxicity. Our hypothesis is that alterations in the redox status of DNA repair proteins may impair DNA damage repair. In order to cause the mild and specific oxidation of protein thiols we employ the disulfide of mercaptoethanol, hydroxy-ethyldisulfide (HEDS). Normal cells are able to prevent thiol-disulfide exchange of their protein and non-protein thiols with HEDS via reducing equivalents produced by the pentose cycle, whose key regulatory enzyme is glucose-6-phosphate-dehydrogenase (G6PD). To prevent this, we investigated a CHO cell line without G6PD activity (E89). Reversibility of observed effects was established by transfecting the G6PD gene back into the E89 mutant. With this model system, we will demonstrate that radiation sensitization, inhibition of DNA repair and inhibition of Ku binding to DNA ends are all caused by incubation of E89 cells with non-toxic concentrations of HEDS. These effects are not seen in parental cells or A1A transfectants. Just as 'p53' may be the 'guardian of the genome' we suggest that G6PD is the 'protector of proteins'. This Application will test this interesting concept using combined biochemical and genetic approaches. Specific Aim 1 will determine the kinetic relationships between HEDS mediated radiosensitization and biochemical modulation. Multiple genetic and biochemical tests will determine the specific sensitivity of Ku to oxidative modification by HEDS treatment. Specific Aim 2 will determine the (bio)chemical mechanism of HEDS oxidation of cellular protein thiols. Specific Aim 3 will investigate other aspects of DNA repair and DNA structural organization to determine their sensitivity to redox regulation.