Repair of oxidative damage in mammalian genomes

修复哺乳动物基因组氧化损伤

• 批准号: 7189356
• 负责人: TAPAS K HAZRA
• 金额: $ 24.02万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7189356
• 关键词: 8-oxoguanine; Address; Affinity; Aging; Antibodies; Base Excision Repairs; Biological Assay; Biology; Cattle; Cell Cycle; Cell Line; Cells; Class; Co-Immunoprecipitations; Complement; Complex; Confocal Microscopy; Cultured Cells; DNA; DNA Adducts; DNA Damage; DNA Repair Pathway; DNA Sequence; DNA glycosylase; DNA lesion; DNA-(apurinic or apyrimidinic site) lyase; DNA-Directed RNA Polymerase; Dependence; Disease; Down-Regulation; ERCC5 gene; Electrophoretic Mobility Shift Assay; Embryo; Enzymatic Biochemistry; Enzyme Kinetics; Escherichia coli; Etiology; Event; Excision; Fatty acid glycerol esters; Fibroblasts; Figs - dietary; Foundations; Frequencies; Future; Gel; Genes; Genetic Engineering; Genetic Transcription; Genome; Genome Stability; Glucocorticoid Receptor; Glucose; Goals; Heterogeneous-Nuclear Ribonucleoprotein U; Homologous Gene; Human; Immunoprecipitation; In Vitro; Ionizing radiation; Kinetics; Knockout Mice; Knowledge; Lead; Left; Length; Lesion; Letters; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measures; Mediating; Milk; Molecular; Mus; Mutagenesis; Mutation; Names; Nuclear; Nuclear Matrix-Associated Proteins; Nucleotide Excision Repair; Null Lymphocytes; OGG1 gene; Organism; Orthologous Gene; Oxidative Stress; PCNA gene; Pathway interactions; Personal Satisfaction; Phase; Phenotype; Phosphoric Monoester Hydrolases; Physiological; Play; Polymerase; Polynucleotide 5' -Hydroxyl-Kinase; Prevention; Process; Protein Chemistry; Proteins; Purines; Pyrimidine; Pyrimidines; RNA Polymerase II; Radiation; Range; Reaction; Reactive Oxygen Species; Recombinant Proteins; Recruitment Activity; Recurrence; Research; Resistance; Roentgen Rays; Role; Sepharose; Small Interfering RNA; Southern Blotting; Staging; Structure; Surgical incisions; System; Techniques; Technology; Testing; Thinking; Transcription-Coupled Repair; Tumor Suppressor Genes; XRCC1 gene; Xeroderma Pigmentosum; base; carcinogenesis; cytosine glycol; dihydrouracil; endonuclease III; glucose oxidase; human APEX1 protein; improved; innovation; insight; mammalian genome; negative elongation factor; novel; oxidation; oxidative DNA damage; plasmid DNA; preference; prevent; purine; repaired; research study; response; thymine glycol; tool

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.

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。在活性氧（ROS）诱导的许多碱基病变中，5-羟基尿嘧啶（5- OHU）和8-氧鸟嘌呤（8-oxoG）被认为是ROS诱导的GC - AT和GC - TA突变的主要原因。在所有生物体中，氧化损伤碱基的修复主要是通过DNA碱基切除修复（BER）途径进行的，该途径由DNA糖基酶的切除启动。只有两种先前被发现的哺乳动物DNA糖基酶，OGG1和NTH1，被认为消除了大部分的氧化损伤。然而，OGG1-和nth1 -缺失小鼠细胞的转录DNA序列缺乏表型和8-oxoG和胸腺嘧啶乙二醇的持续修复表明存在额外的糖基化酶。我们最近发现并部分鉴定了大肠杆菌Nei的两个人类同源基因，并将它们命名为NElL (Nei-like)-1和NElL (Nei-like)- 2。它们的独特之处在于，不像大多数只对双链DNA有活性的DNA糖基酶，NEILs从气泡DNA中去除病变。NEIL2对泡DNA中5-OHU的亲和力和活性比双工DNA高4- 5倍，它从泡DNA中切除8-oxoG，而不从双工DNA中切除。这些观察结果强烈表明，NEIL2在转录泡损伤修复中起主要作用，因此有助于在OGG1-null细胞中观察到的转录偶联碱基切除修复（TC-BER）。NEIL2与RNA聚合酶II （Pol II）和异质核核糖核蛋白（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设一致，hnRNP-U作为一种丰富的多功能核基质蛋白，也被证明具有调节转录的作用。NEIL2像大肠杆菌MutM/Nei一样进行β - δ反应，产生3'-P，该3'-P被多核苷酸激酶（PNK）去除，但不被ap内切酶去除。NEIL2对气泡DNA的强烈偏好，以及它与Pol II和hnRNP-U的关联，以及一种不依赖于ape1但依赖于pnk的新型修复途径的发现，为全面了解5- OHU、8-oxoG和其他致突变性病变如何在基因组的转录区和非转录区修复，以及NEIL2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。包括重组DNA技术、酶动力学、检测蛋白-蛋白相互作用的共免疫沉淀实验以及siRNA技术在内的多种技术将被用于实现以下目标：(1)阐明气泡DNA中hnRNP-U刺激NEIL2活性的机制基础；(2)验证neil2介导的链切割导致转录阻滞的假设；(3)验证NEIL2参与pnk依赖性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，这将为氧化碱基的基因毒性作用的细胞反应提供明确的见解，并有助于制定更好的预防或治疗辐射和ros诱导的癌变的策略。