Repair of oxidative damage in mammalian genomes

描述（由申请人提供）：氧化诱导的DNA损伤与许多疾病（包括癌症）的病因学和衰老有关。在由活性氧（ROS）诱导的许多碱基损伤中，5-羟基尿嘧啶（5- OHU）和8-氧代鸟嘌呤（8-oxoG）被认为分别负责大多数ROS诱导的GC至AT和GC至TA突变。所有生物体中氧化损伤碱基的修复主要通过DNA碱基切除修复（BER）途径发生，由DNA糖基化酶切除起始。只有两个先前表征的哺乳动物DNA糖基化酶，OGG 1和NTH 1，被认为切除大部分的氧化损伤。然而，缺乏表型和持续修复的8-oxoG和胸腺嘧啶乙二醇从转录的DNA序列的OGG 1和NTH 1无效的小鼠细胞表明存在额外的糖基化酶。我们最近发现并部分表征了大肠杆菌Nei的两个人类直系同源物，并将其命名为NEIL（Nei-like）-1和2。它们的独特之处在于，与仅对双链DNA有活性的大多数DNA糖基化酶不同，NEIL从气泡DNA中切除病变。NEIL 2对气泡中的5-OHU具有比双链体DNA高4至5倍的亲和力和活性，并且它从气泡中切除8-oxoG而不是双链体DNA。这些观察结果强烈表明，NEIL 2在修复转录泡中的损伤中起着重要作用，因此有助于在OGG 1缺失细胞中观察到的转录偶联碱基切除修复（TC-BER）。NEIL 2与RNA聚合酶II（Pol II）和异质性核核糖核蛋白（hnRNP-U）的稳定相互作用以及NEIL 2缺陷细胞中内源性突变的显著增加与这一假设一致。NEIL 2与E. coli MutM/Nei，产生3 '-P，其被多核苷酸激酶（PNK）去除，但不被AP-内切酶去除。NEIL 2对气泡DNA的强烈偏好及其与Pol II和hnRNP-U的关联以及一种不依赖APE 1但依赖PNK的新修复途径的发现，为全面了解5- OHU、8-oxoG和其他诱变损伤如何在基因组的转录与非转录区域中修复奠定了基础，以及NEIL 2介导的修复如何涉及与hnRNP-U和Pol II的特异性相互作用。本研究将采用多种技术，包括重组DNA技术、酶动力学、检测蛋白质-蛋白质相互作用的免疫共沉淀实验以及siRNA技术，来实现以下目标：（1）阐明气泡DNA中hnRNP-U刺激NEIL 2活性的机制基础;（2）检验NEIL 2介导的链切割导致转录停滞的假说;以及（3）检验NEIL 2参与PNK依赖的TC-BER的假设。我们研究的长期目标是阐明氧化碱基的TC-BER的机制基础，这应该为细胞对氧化碱基的遗传毒性效应的反应提供明确的见解，并有助于制定更好地预防或治疗辐射和ROS诱导的致癌作用的策略。

DESCRIPTION (provided by applicant): Oxidatively induced DNA lesions have been implicated in the etiology of many diseases (including cancer), and in aging. Among the many base lesions induced by reactive oxygen species (ROS), 5-hydroxyuracil (5- OHU) and 8-oxoguanine (8-oxoG) are thought to be responsible for the majority of ROS-induced GC to AT and GC to TA mutations, respectively. Repair of oxidatively damaged bases in all organisms occurs primarily via the DNA base excision repair (BER) pathway, initiated with excision by DNA glycosylases. Only two previously characterized mammalian DNA glycosylases, OGG1 and NTH1, are thought to excise most of the oxidative damage. However, the lack of phenotype and continued repair of 8-oxoG and thymine glycol from the transcribed DNA sequences of OGG1- and NTH1-null mouse cells suggested the presence of additional glycosylases. We have recently discovered and partially characterized two human orthologs of E.coli Nei, and named them NElL (Nei-like)-1 and 2. They are unique in that, unlike most DNA glycosylases which are active only with duplex DNA, NEILs excise lesions from bubble DNA. NEIL2 has a 4- to 5-fold higher affinity and activity for 5-OHU in bubble vs duplex DNA, and it excises 8-oxoG from bubble but not duplex DNA. These observations strongly suggest that NEIL2 plays a major role in repairing lesions in transcription bubbles, and so contributes to the transcription-coupled base excision repair (TC-BER) observed in OGG1-null cells. The stable interaction of NEIL2 with RNA Polymerase II (Pol II) and heterogeneous nuclear ribonucleo protein (hnRNP-U), and the significant increase in endogenous mutations in NEIL2-deficient cells, are consistent with this hypothesis, hnRNP-U, an abundant multifunctional nuclear matrix protein, has also been shown to regulate transcription. NEIL2 carries out a beta-delta-reaction like E. coli MutM/Nei, generating a 3'-P which is removed by polynucleotide kinase (PNK), but not by AP-endonuclease. The discovery of NEIL2's strong preference for bubble DNA, and its association with Pol II and hnRNP-U and a novel repair pathway that is APE1-independent but PNK-dependent, have set the stage for developing a comprehensive picture of how 5- OHU, 8-oxoG and other mutagenic lesions, are repaired in transcribed vs. nontranscribed regions of the genome, and how NEIL2-mediated repair involves specific interactions with hnRNP-U and Pol II. A variety of techniques, including recombinant DNA technology, enzyme kinetics, coimmunoprecipitation experiments to examine protein-protein irteractions, and finally siRNA technology, will be used to pursue the following aims, to: (1) elucidate the mechanistic basis by which NEIL2's activity is stimulated by hnRNP-U in bubble DNA; (2) test the hypothesis that NEIL2-mediated strand incision leads to transcriptional arrest; and (3) test the hypothesis that NEIL2 is involved in PNK-dependent TC-BER. The long-term goal of our research is to elucidate the mechanistic basis of TC-BER of oxidized bases, which should provide definitive insights into cellular responses to the genotoxic effects of the oxidized bases and help develop strategies for better prevention or treatment of radiation- and ROS-induced carcinogenesis.