Repair of oxidative damage in mammalian genomes

描述(申请人提供)：氧化诱导的DNA损伤与许多疾病(包括癌症)的病因学有关，也与衰老有关。在ROS引起的许多碱基损伤中，5-羟基尿嘧啶(5-OHU)和8-氧鸟嘌呤(8-oxoG)分别被认为是ROS诱导的GC到AT和GC到TA突变的主要原因。在所有生物体中，氧化损伤的碱基修复主要通过DNA碱基切除修复(BER)途径进行，该途径由DNA糖基酶切除启动。只有两种先前表征的哺乳动物DNA糖基酶，OGG1和NTH1，被认为可以切除大部分氧化损伤。然而，从OGG1和NTH1缺失的小鼠细胞的转录DNA序列中缺乏表型和8-oxoG和胸腺嘧啶二醇的持续修复表明存在额外的糖基酶。我们最近发现并部分鉴定了E.ColiNei的两个人类同源物，并将它们命名为Nell(Nei-like)-1和2。它们的独特之处在于，不像大多数DNA糖基酶只在双链DNA上活性，Neils从气泡DNA中切除病变。NEIL2对泡泡中5-OHU的亲和力和活性是双链DNA的4~5倍，并且它能从泡泡中去除8-oxoG，但不能从双链DNA中去除8-oxoG。这些观察有力地表明，NEIL2在修复转录泡中的损伤方面发挥了重要作用，因此有助于在OGG1缺失细胞中观察到转录偶联碱基切除修复(TC-BER)。NEIL2与RNA聚合酶II(Pol II)和异质性核糖核蛋白(hnRNP-U)的稳定相互作用，以及NEIL2缺陷细胞内源性突变的显著增加，与这一假说相一致，hnRNP-U是一种丰富的多功能核基质蛋白，也被证明调控转录。NEIL2类似于E.coliMutM/NeI进行β-增量反应，产生一个可被多核苷酸激酶(PNK)去除的3‘-P，但不能被AP-内切酶去除。Neil 2的S发现的对泡泡DNA的强烈偏好，及其与Pol II和hnRNP-U的关联，以及一种不依赖于APE 1但依赖于PNK的新的修复途径，为全面了解5-OHU，8-oxoG和其他突变损伤是如何在基因组的转录和非转录区域修复，以及NIL-2介导的修复如何涉及与hnRNP-U和Pol II的特定相互作用奠定了基础。目的：(1)阐明泡状DNA中hnRNP-U刺激NIL-2‘S活性的机制；(2)检验NEIL2介导的链切割导致转录停滞的假设；(3)检验NEIL2参与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TC-BER的机制基础，这将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.