Oxidative DNA Damage and the Analysis of 8-Oxyog Repair

DNA 氧化损伤和 8-Oxyog 修复分析

• 批准号: 7046153
• 负责人: Walter Andy Deutsch
• 金额: $ 30.84万
• 依托单位: LSU PENNINGTON BIOMEDICAL RESEARCH CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7046153
• 关键词: DNA; DNA damage; DNA repair; RNA interference; chemical binding; free radical oxygen; gel mobility shift assay; gene expression; guanine; human genetic material tag; immunoprecipitation; mutant; oxidative stress; protein binding; protein localization; protein protein interaction; protein structure function; ribosomal proteins; site directed mutagenesis; small interfering RNA; surface plasmon resonance; tissue /cell culture

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.

描述（由申请人提供）：DNA中7，8-二氢-8-氧代鸟嘌呤（8-oxoG）的形成主要是通过活性氧（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 -> T颠换突变，从而使细胞易患多种疾病，如癌症。然而，所有生物体都有能力通过碱基切除修复（BER）去除这种损伤，其中该途径的第一步是通过N-糖基化酶活性释放8-oxoG。在许多情况下，N-糖基化酶具有固有的AP裂解酶活性，其随后裂解脱碱基位点，脱碱基位点又可以根据所涉及的糖基化酶进行δ-消除。我们以前在果蝇中的研究发现，核糖体蛋白S3（dS 3）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dS 3转化为类似于人S3（hS 3）的活性，因为它仅具有AP裂解酶活性。对hS 3的后续研究已经鉴定了hS 3对8-oxG的非常高的结合亲和力，如通过表面等离子体共振（SPR）所揭示的，即使hS 3缺乏N-糖基化酶活性。同样的SPR技术也有助于显示hS 3与人BER蛋白8-oxoG糖基化酶（OGG 1）和APE/ref-1的正相互作用。这些综合结果制定了竞争性更新的基础，我们希望深入研究hS 3在BER中的参与。有三个目标。前两个致力于通过定点诱变去除DNA结合和蛋白质：蛋白质相互作用结构域。用于确定hS 3作用的重要体外试验将是通过在重构BER测定中使用野生型和突变体形式。第三个目标将集中在hS 3在体内过表达和低表达的生物学后果。使用野生型和dS 3突变体的细胞存活率、诱变和亚细胞位置的变化应产生定义S3在碱基切除修复中的作用的结果。我们的预期是，一旦hS 3与8-oxoG结合，它将对BER产生不利影响，这也许可以解释为什么某些组织含有大量的8-oxoG。相反，hS 3可以作为OGG 1和APE/ref-1的支架，从而对BER产生积极的结果。成功完成本申请中提出的目标应有助于阐明这两种情况。