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.

氧化损伤主要通过碱基切除修复（BER）途径从DNA中去除。BER通过四个酶促步骤进行，但现在清楚的是，其他几种蛋白质通过蛋白质-蛋白质相互作用调节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的和功能性的，并且共同支持“接力棒传递”模型，其中BER在不同的步骤中发生，这些步骤由作为修复复合物的组分的个体蛋白质相互作用支持，可能位于DNA损伤处。有趣的是，这些包括两种与过早衰老疾病相关的蛋白质，Cockayne综合征（CS）互补组B基因（CS B）和Werner综合征基因（WRN）。在CS细胞中，核和线粒体DNA中的氧化DNA损伤修复存在缺陷，这可能是疾病的主要潜在原因。我们发现，CSB缺陷的细胞积累氧化的碱基，8-羟基鸟嘌呤和8-羟基腺嘌呤，氧化应激后，与观察到的CSB和氧代鸟嘌呤DNA糖基化酶（OGG 1），主要的DNA糖基化酶8-oxoG修复，在体内是一个复杂的一致。我们还发现CSB蛋白与Nei样DNA糖基化酶NEIL 1发生物理相互作用，NEIL 1也参与氧化碱基的修复。这种相互作用显着刺激NEIL 1的催化活性，包括糖基化酶和AP裂合酶。观察到CSB缺陷小鼠在脑组织中积累了显著更高水平的几种氧化DNA碱基，包括fapyadenine和fapyguanine，这支持了CSB蛋白在体内去除氧化损伤中的作用。值得一提的是，FRES 1损伤被认为是NEIL 1的典型底物，强调了这种蛋白相互作用的生物学相关性。 我们最近证明，CSB蛋白还与PARP 1相互作用，PARP 1是一种参与单链断裂修复早期步骤的蛋白质，并且这两种蛋白质在细胞对氧化应激的反应中相互作用。CSB是PARP-1核糖基化的底物，并且这两种蛋白质可能在碱基切除过程中一起起作用。我们的研究结果表明，CSB蛋白在氧化性DNA损伤的修复中起着重要作用，并且未修复病变的积累，特别是在靶组织中，如大脑，可能与CS病理学有关，其特征在于严重的早发性神经变性。 此外，我们已经确定了一种新的催化活性的CSB蛋白。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的唯一鉴定的催化活性是ATP水解。我们发现CSB有效地催化两条互补DNA链的退火。我们现在正在绘制这种新的活动，以更好地了解其生物相关性。 8-oxoG的修复是特别感兴趣的，因为这种病变被认为是高度致突变的，并随着年龄的增长而积累。我们发现OGG 1与细胞周期蛋白依赖性激酶cdk 4相互作用并被其磷酸化。这种翻译后修饰调节OGG 1的催化活性，表明信号通路在氧化DNA损伤反应中的作用。我们正在研究OGG 1的其他蛋白质相互作用，以了解如何在体内调节氧化损伤的修复。我们发现OGG 1也与重组蛋白RAD 52相互作用，这表明这两种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的相互作用，其中RAD 52刺激OGG 1的催化活性和OGG 1抑制RAD 52催化的DNA链退火和入侵。此外，OGG 1和RAD 52之间的物理相互作用在暴露于氧化应激的细胞中增加，表明这种相互作用在细胞对氧化DNA损伤的反应中是重要的。 我们最近已经表明，WRN蛋白也与几种BER蛋白，包括聚合酶B，瓣状核酸内切酶1（FEN-1），AP核酸内切酶（APE）和NEIL-1的物理和功能相互作用。我们发现WRN强烈刺激FEN-1切割、pol B链置换活性和NEIL 1糖基化酶活性。WRN在BER中的作用的进一步支持来自我们的观察，即至少三种不同的氧化损伤，8-oxoG，FapyG和FapyA的水平在来自WRN缺陷细胞的DNA中显著升高，并且在来自WRN表达被RNAi降低的细胞的提取物中长斑BER活性降低。我们的研究结果支持WRN通过刺激pol B链置换促进长补丁BER的模型。