Oxidative DNA Damage and the Analysis of 8-Oxyog Repair

描述(申请人提供)：DNA中7，8-二氢-8-氧鸟嘌呤(8-oxoG)的形成主要是通过活性氧物种(ROS)的作用。DNA中8-oxoG的存在有可能在DNA复制过程中与A错配，导致G-&gt；T颠倒突变，从而使细胞容易患上癌症等多种疾病。然而，所有的生物都有能力通过碱基切除修复(BER)来去除这种损伤，这一途径的第一步是通过N-糖基酶活性释放8-oxoG。在许多情况下，N-糖基酶具有内在的AP裂解酶活性，该活性随后裂解碱性部位，根据所涉及的糖基酶的不同，该碱性部位又可能受到增量消除的影响。我们以前对果蝇的研究发现，核糖体蛋白S3(DS3)具有所有这三种活性。值得注意的是，通过单个氨基酸的改变，我们能够将DS3转化为类似于人类S3(HS3)的活性，因为它只具有AP裂解酶活性。后来对HS3的研究发现，尽管HS3缺乏N-糖基酶活性，但表面等离子体共振(SPR)显示HS3与8-Oxg有很高的结合亲和力。同样的SPR技术也有助于表明HS3与人类BER蛋白8-oxoG糖基酶(OGG1)和APE/REF-1正相互作用。这些综合结果形成了这次竞争更新的基础，我们希望在此基础上深入研究HS3在BER中的参与。有三个目标。前两个致力于通过定点突变去除DNA结合和蛋白质：蛋白质相互作用结构域。确定HS3作用的一个重要的体外试验将是在重组的误码率分析中使用野生型和突变型。第三个目标将集中在体内HS3过度表达和低表达的生物学后果。使用野生型和ds3突变体在细胞存活、突变和亚细胞定位方面的变化应该会产生一个确定S3在碱基切除修复中的作用的结果。我们的预期是，HS3一旦与8-oxoG结合，将对误码率产生不利影响，这可能解释了为什么一些组织含有大量的8-oxoG。相反，HS3可能作为OGG1和APE/REF-1的支架，从而在误码率方面产生积极的结果。本申请中提出的目标的成功完成应该会对这两种情况有所启示。

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.