Oxidative DNA Damage And Its Processing

氧化损伤主要通过碱基切除修复(BER)途径从DNA中移除。BER通过四个酶步骤进行，但现在已经清楚，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和功能的，共同支持“传递接力棒”模型，在该模型中，BER在不同步骤中发生，由单个蛋白质相互作用支持，这些蛋白质相互作用是修复复合体的组成部分，可能位于DNA损伤处。有趣的是，这些基因包括两种与早衰障碍相关的蛋白质，Cockayne综合征(CS)互补B组基因(CSB)和Werner综合征基因(WRN)。在CS细胞中，核和线粒体DNA氧化损伤的修复存在缺陷，这可能是该病的主要潜在原因。我们发现CSB缺陷细胞在氧化应激后积累氧化碱基8-羟基鸟嘌呤和8-羟基腺嘌呤，这与观察到CSB和8-oxoG修复的主要DNA糖基酶OGG1在体内处于一个复合体中是一致的。我们还发现CSB蛋白与Nei样DNA糖基酶NEIL1物理上相互作用，NEIL1也参与氧化碱基的修复。这种相互作用显著地刺激了NEIL1的催化活性，既包括糖基酶，也包括解酶。观察到CSB缺陷小鼠在脑组织中积累了显著较高水平的几种氧化DNA碱基，包括法比腺嘌呤和法比鸟嘌呤，这支持了CSB蛋白在体内清除氧化损伤中的作用。值得注意的是，Fapy损伤被认为是NEIL1的典型底物，强调了这种蛋白质相互作用的生物学相关性。 我们最近证明了CSB蛋白也与PARP1蛋白相互作用，PARP1蛋白参与单链断裂修复的早期步骤，并且这两种蛋白在细胞对氧化应激的反应中相互作用。CSB是PARP-1核糖化的底物，这两种蛋白很可能在碱基切除过程中共同发挥作用。我们的结果表明，CSB蛋白在DNA氧化损伤的修复中起着重要作用，未修复损伤的积累，特别是在靶组织，如脑，可能与以严重的早发性神经变性为特征的CS病理有关。 此外，我们还鉴定了CSB蛋白的一种新的催化活性。尽管CSB有7个保守的解旋酶结构域(SWI/SNF蛋白家族的特征)，但唯一发现的催化活性是ATP水解酶。我们发现CSB有效地催化了两条互补的DNA链的退火。我们现在正在绘制这一新的活动图，以更好地了解它的生物学相关性。 8-oxoG的修复特别有意义，因为这种损伤被认为是高度突变的，并随着年龄的增长而积累。我们发现OGG1与细胞周期蛋白依赖性蛋白依赖性蛋白激酶CDK4相互作用并可被其磷酸化。这种翻译后修饰调节了OGG1的催化活性，表明信号通路在对DNA氧化损伤的反应中发挥了作用。我们正在研究OGG1的其他蛋白质相互作用，以了解氧化损伤的修复在体内是如何调控的。我们发现OGG1也与重组蛋白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功能上的相互作用，其中RAD52刺激OGG1的催化活性，而OGG1抑制RAD52催化的DNA链的退火和侵袭。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损伤的反应中是重要的。 我们最近发现，WRN蛋白还与几种BER蛋白在物理和功能上相互作用，包括聚合酶b、Fen-1、AP内切酶(APE)和NeIL-1。我们发现WRN强烈地刺激FEN-1切割、Polb链置换活性和NEIL-1糖基酶活性。进一步支持WRN在BER中的作用来自我们的观察，即在WRN缺陷细胞的DNA中，至少三种不同的氧化损伤，8-oxoG，FapyG和FapyA的水平显著升高，而在WRN表达被RNAi下调的细胞提取物中，长斑块BER活性降低。我们的结果支持一个模型，在该模型中，WRN通过刺激Polb链移动来促进长斑块BER。

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.