Oxidative DNA Damage And Its Processing

DNA氧化损伤及其处理

• 批准号: 7592041
• 负责人: Vilhelm A Bohr
• 金额: $ 65.4万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592041
• 关键词: 8-hydroxyadenine; 8-hydroxyguanine; 8-hydroxyguanosine; ATP Hydrolysis; Age; Aging; Aging-Related Process; Base Excision Repairs; Biological; Brain; Cell Extracts; Cells; Characteristics; Cockayne Syndrome; Complex; Cyclin-Dependent Kinase 4; DNA; DNA Excision Repair Protein ERCC-6; DNA Modification Process; DNA glycosylase; DNA lesion; DNA-(apurinic or apyrimidinic site) lyase; Development; Disease; Drosophila snf protein; ERCC6 gene; Excision; Functional disorder; Gene Expression; Gene Proteins; Genes; Genetic Recombination; Genomic Instability; Goals; Individual; Lead; Lesion; Life; Lipids; Lyase; Malignant Neoplasms; Maps; Metabolism; Mitochondrial DNA; Modeling; Modification; Mus; Mutagenesis; Mutate; Nerve Degeneration; Nuclear; OGG1 gene; Organism; Oxidative Stress; Pathology; Pathway interactions; Play; Polymerase; Post-Translational Protein Processing; Premature aging syndrome; Process; Protein Family; Proteins; RNA Interference; RTH-1 Nuclease; Repair Complex; Role; Signal Pathway; Single Strand Break Repair; Site; Surgical incisions; Thinking; Tissues; Werner Syndrome; base; brain tissue; early onset; environmental agent; helicase; in vivo; interest; novel; oxidative DNA damage; pol genes; protein function; protein protein interaction; repair enzyme; repaired; response

Oxidative lesions are removed from DNA primarily via the base excision repair (BER) pathway. BER is carried out through four enzymatic steps, but is now clear that several other proteins modulate BER efficiency through protein-protein interactions. We and others identified several protein interactions for the core BER enzymes. These protein interactions are physical and functional and together support the "passing of baton" model, in which BER takes place in different steps supported by individual protein interactions that are components of a repair complex, possibly situated at the DNA lesion. Interestingly, these include two proteins associated with premature aging disorders, the Cockayne syndrome (CS) complementation group B gene (CSB) and the Werner syndrome gene (WRN). In CS cells, there are deficiencies in the repair of oxidative DNA damage in the nuclear and mitochondrial DNA, and this may be a major underlying cause of the disease. We found that CSB-deficient cells accumulate oxidized bases, 8-hydroxyguanine and 8-hydroxyadenine, after oxidative stress, consistent with the observation that CSB and oxoguanine DNA glycosylase (OGG1), the major DNA glycosylase for 8-oxoG repair, are in a complex in vivo. We also found that the CSB protein physically interacts with the Nei-like DNA glycosylase, NEIL1, which is also involved in the repair of oxidized bases. This interaction significantly stimulates NEIL1 catalytic activities, both the glycosylase as well as the AP-lyase. The observation that CSB-deficient mice accumulate significantly higher levels of several oxidized DNA bases in brain tissue, including fapyadenine and fapyguanine, supports a role for the CSB protein in the removal of oxidized lesions in vivo. It is notewhorty that Fapy lesions are considered canonical substrates for NEIL1, underscoring the biolgical relevance for this protein interaction. We recently demonstrated that the CSB protein also interacts with PARP1, a protein involved in the early steps of single-strand break repair, and that these two proteins cooperate in the cellular responses to oxidative stress. CSB is a substrate for PARP-1 ribosylation and it is likely that these two proteins function together in the process of base excision. Our results indicate that the CSB protein plays an important role in the repair of oxidative DNA damage and that accumulation of unrepaired lesions, particular in target tissues, like the brain, may be relevant to the CS pathology, which is characterized by severe early onset neurodegeneration. Moreover, we have identified a novel catalytic activity of the CSB protein. Despite having 7 conserved helicase domains (characteristic of the SWI/SNF protein family), the only identified catalytic activity of CSB was ATP hydrolysis. We found that CSB efficiently catalyzes the annealing of two complementary strands of DNA. We are now mapping this novel activity to gain a better understanding of its biological relevance. Repair of 8-oxoG is of special interest since this lesion is believed to be highly mutagenic and accumulates with age. We find that OGG1 interacts with and can be phosphorylated by the cyclin-dependent kinase cdk4. This post-translational modification modulates OGG1 catalytic activity, suggesting a role for signaling pathways in the response to oxidative DNA damage. We are studying other protein interactions of OGG1 in order to understand how repair of oxidative lesions is regulated in vivo. We find that OGG1 also interacts with the recombination protein RAD52, suggesting a possible interplay between these two repair pathways. We find a reciprocal functional interaction between these two proteins, in which RAD52 stimulates OGG1 catalytic activity and OGG1 inhibits RAD52-catalysed DNA strand annealing and invasion. Moreover, the physical interaction between OGG1 and RAD52 increases in cells exposed to oxidative stress, indicating that this interaction is important in the cellular response to oxidative DNA damage. We have recently shown that the WRN protein also interacts physically and functionally with several BER proteins, including polymerase b, flap endonuclease 1 (FEN-1), AP endonuclease (APE) and NEIL-1. We find that WRN strongly stimulates FEN-1 incision, pol b strand displacement activity and NEIL1 glycosylase activity. Further support for a role of WRN in BER comes from our observation that the levels of at least three different oxidative lesions, 8-oxoG, FapyG and FapyA, are significantly elevated in DNA from WRN-deficient cells and that long-patch BER activity is decreased in extracts from cells in which WRN expression is decreased by RNAi. Our results support a model in which WRN promotes long-patch BER by stimulating pol b strand displacement.

氧化病变主要通过碱基切除修复（BER）途径从DNA中去除。 BER是通过四个酶促步骤进行的，但现在很明显其他几种蛋白质通过蛋白质 - 蛋白质相互作用来调节BER效率。我们和其他人确定了核心BER酶的几种蛋白质相互作用。这些蛋白质相互作用是物理和功能性的，并共同支持“指挥棒的传递”模型，其中BER发生在不同的步骤中，由单个蛋白质相互作用支持，这些相互作用是修复复合物的组成部分，可能位于DNA病变位于DNA病变。有趣的是，这些蛋白包括与早衰疾病相关的两种蛋白质，即Cockayne综合征（CS）补充B基因（CSB）和Werner综合征基因（WRN）。在CS细胞中，修复核和线粒体DNA中氧化DNA损伤的缺乏，这可能是该疾病的主要根本原因。我们发现，CSB缺乏的细胞在氧化应激之后积累了氧化的碱，8-羟基鸟嘌呤和8-羟基苯胺，这与观察到的观察到CSB和Oxoguanine DNA糖基酶（OGG1）（OGG1），主要的DNA糖基酶的主要DNA糖基酶，用于8-氧化物的主要DNA糖基酶。我们还发现，CSB蛋白与Nei样DNA糖基化酶Neil1物理相互作用，该糖基酶也参与了氧化碱基的修复。这种相互作用显着刺激了Neil1催化活性，包括糖基化酶和AP-乙醇酶。 CSB缺陷型小鼠在脑组织中积累了几种氧化DNA碱的水平显着较高，包括fapaighadenine和fapyguanine，支持CSB蛋白在体生中去除氧化病变中的作用。值得注意的是，Fapy病变被认为是Neil1的规范底物，强调了这种蛋白质相互作用的生物学相关性。 我们最近证明，CSB蛋白还与PARP1相互作用，PARP1是一种参与单链断裂修复的早期步骤的蛋白质，并且这两种蛋白质在细胞对氧化应激的反应中配合。 CSB是PARP-1核糖基化的底物，这两种蛋白可能在碱性切除过程中起作用。我们的结果表明，CSB蛋白在氧化DNA损伤的修复中起着重要作用，并且在靶向组织中，像大脑这样的靶组织中的未经修复病变的积累可能与CS病理有关，CS病理与CS病理有关，该病理的特征是严重的早期发作神经变性。 此外，我们已经确定了CSB蛋白的新型催化活性。尽管具有7个保守的解旋酶结构域（SWI/SNF蛋白家族的特征），但CSB唯一鉴定出的催化活性是ATP水解。我们发现CSB有效地催化了两种互补的DNA链的退火。现在，我们正在绘制这项新型活动，以更好地了解其生物学相关性。 8-oxog的修复是特别感兴趣的，因为这种病变被认为是高度诱变的，并且随着年龄的增长而积聚。我们发现OGG1与细胞周期蛋白依赖性激酶CDK4相互作用并可以磷酸化。这种翻译后的修饰调节OGG1催化活性，这表明信号通路在对氧化DNA损伤响应中的作用。我们正在研究OGG1的其他蛋白质相互作用，以了解如何在体内调节氧化病变的修复。我们发现OGG1还与重组蛋白RAD52相互作用，这表明这两个修复途径之间可能存在相互作用。我们发现这两种蛋白质之间存在相互的功能相互作用，其中RAD52刺激OGG1催化活性，而OGG1抑制RAD52催化的DNA链DNA链退火和侵袭。此外，在暴露于氧化应激的细胞中，OGG1和RAD52之间的物理相互作用增加，表明这种相互作用在细胞对氧化DNA损伤的反应中很重要。 我们最近表明，WRN蛋白还与多种BER蛋白在物理和功能上相互作用，包括聚合酶B，皮瓣内核酸酶1（FEN-1），AP内核酸酶（APE）和Neil-1。我们发现WRN强烈刺激FEN-1切口，Pol B链位移活性和NEIL1糖基化酶活性。我们观察到，对WRN在BER中的作用的进一步支持是，至少三种不同的氧化病变的水平8-oxog，Fapyg和Fapya的水平显着升高，从WRN缺陷型细胞中的DNA中显着升高，并且从RNAI降低WRN表达的提取物中，提取物中的长距离BER活性降低。我们的结果支持了一个模型，其中WR通过刺激Pol b链位移来促进长块ber。