The role of the Cockayne syndrome proetin

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。以前我们发现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。 此前，我们证明了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，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。 为了进一步探讨CSB在线粒体中的作用，我们评估了氧化应激后CSB的线粒体定位。我们发现，甲萘二酮治疗后CSB对线粒体的定位增加，这会导致一种形式的氧化应激。此外，我们发现与野生型细胞相比，CSB缺陷细胞中8-氧鸟嘌呤、尿嘧啶和5-羟基尿嘧啶的切割活性降低。这种缺陷与线粒体内膜相关的BER活性丧失有关。这些依赖于CSB的改变具有功能后果，因为我们在CSB缺陷细胞中观察到mtDNA突变增加。综上所述，结果表明CSB通过帮助招募、稳定和/或保留修复复合体中与内膜相关的修复复合体中的BER蛋白，在线粒体BER中发挥直接作用。

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. Previously 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. Previously we 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. To further explore the role of CSB in mitochondria, we evaluated the mitochondrial localization of CSB following oxidative stress. We found increased CSB localization to mitochondria following menadione treatment, which causes a form of oxidative stress. Additionally, we found reduced 8-oxo-guanine, uracil, and 5-hydroxy-uracil incision activities in CSB-deficient cells compared to wild-type cells. This deficiency correlated with a loss of mitochondrial inner membrane associated BER activities. These CSB-dependent changes had a functional consequence because we observed elevated mtDNA mutations in CSB deficient cells. Together the results suggest that CSB plays a direct role in mitochondrial BER by helping to recruit, stabilize, and/or retain BER proteins in repair complexes that in mitochondria are associated with the inner membrane.