Oxidative DNA Damage and the Analysis of 8-Oxyog Repair

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DESCRIPTION (provided by applicant): The formation of 7, 8-dihydo-8-oxoguanine (8-oxoG) in DNA is primarily through the action of reactive oxygen species (ROS). The presence of 8-oxoG in DNA has the potential to mispair with A during DNA replication, leading to G -> T transversion mutations that can predispose cells for a number of disease states such as cancer. All organisms, however, have the ability to remove this lesion via Base Excision Repair (BER), in which the first step in this pathway is the liberation of 8-oxoG by an N-glycosylase activity. In many instances N-glycosylases possess an intrinsic AP lyase activity that subsequently cleaves the abasic site, which in turn may be subject to delta-elimination depending upon the glycosylase involved. Our previous studies in Drosophila found that the ribosomal protein S3 (dS3) possessed all three of these activities. Notably, through a single amino acid change we were able to convert dS3 into an activity that resembled human S3 (hS3) in that it only possessed AP lyase activity. Subsequent studies on hS3 have identified a very high binding affinity for hS3 towards 8-oxG as revealed by surface plasmon resonance (SPR), even though hS3 lacks N-glycosylase activity. The same SPR technology was also instrumental in showing that hS3 positively interacts with the human BER proteins 8-oxoG glycosylase (OGG1) and APE/ref-1. These combined results have formulated the basis of this competitive renewal, in which we wish to examine in depth the involvement of hS3 in BER. There are three aims. The first two are devoted to removing the DNA binding and protein:protein interaction domains through site-directed mutagenesis. An important in vitro test for establishing the role of hS3 will be through the use of wild-type and mutant forms in reconstituted BER assays. The third aim will concentrate on the biological consequences of in vivo overexpression and underexpression of hS3. Changes in cell survival, mutagenesis, and subcellular location using wild-type and dS3 mutants should produce an outcome that defines the role of S3 in base excision repair. Our expectation is that hS3 will adversely impact BER once it binds to 8-oxoG, explaining perhaps why some tissues harbor high amounts of 8-oxoG. Conversely, hS3 may act as a scaffold for OGG1 and APE/ref-1, thereby producing a positive outcome on BER. The successful completion of the aims proposed in this application should shed light on both of these scenarios.