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Base Excision Repair (BER) and Nucleotide Excision Repair (NER) of both natural and synthetic nucleotides.

机译：天然和合成核苷酸的碱基切除修复（BER）和核苷酸切除修复（NER）。

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摘要

DNA repair glycosylases locate and excise damaged bases from DNA during the first step of BER, playing central roles in the preservation of the genome and the prevention of disease. The Escherichia coli ( E. coli) glycosylases Fpg, MutY, and the human glycosylase hOGG1 are involved in the repair of the mutagenic oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (8-OxoG or OG). To investigate the required features utilized for damage recognition and catalysis, a series of substituted indole nucleotides that mimic features of OG were tested as direct substrates for these glycosylases. Results demonstrate that the OG glycosylases hOGG1 and in particular, Fpg, can recognize and cleave non-polar OG mimics, despite their lack of hydrogen bonding groups and basic atoms. In contrast to Fpg and hOGG1, single turnover kinetic experiments conducted with MutY and the nonpolar OG analogs based paired to A resulted in no detectable A excision. The relative rates of base excision chemistry of the nonpolar analogs displayed by each glycosylase highlights key differences in the mechanisms of recognition and catalysis employed by Fpg, hOGG1, and MutY.;There are various diseases associated with defects in DNA repair. One such example is an inherited form of colon cancer, referred to as MUTYH-associated polyposis (MAP). MAP is caused by defects in the human homologue of MutY (MUTYH), which works to prevent G to T transversion mutations associated with the OG lesion. MutY is a unique glycosylase, which detects and removes the normal adenine when mispaired across from OG. To evaluate which interactions between MutY and the OG:A substrate are vital, a series of OG and adenine nucleotide analogs which vary from the natural substrate in the base pair stability, hydrogen bonding capability, and glycosidic bond stability were chosen. To determine which part of the MutY reaction pathway these substrate modifications affect, the binding affinity, the rate of catalysis, and the overall cellular repair were all measured. Results reported in this dissertation support previous observations demonstrating that any structural deviation from OG dramatically reduces binding, efficient catalysis in vitro, and successful repair in bacterial cells, demonstrating the critical importance of precise recognition of OG for efficient MutY activity. Interestingly, only modifications of adenine at the C-2 and N-1 positions where shown to negatively affect OG:A cellular repair. This suggests that efficient binding and modest catalytic activity are not the only requirements for MutY activity and underscores the importance of confirmation of adenine, in addition to the identification of OG in an OG:A mismatch.;Further oxidation of OG results in the production of the highly mutagenic hydantoin lesions, guanidinohydantion (Gh) and spiroiminodihydantoin (Sp). Interestingly, oxidation of OG in the presence of primary amines results in the formation of hydantoin amine adducts. The hydantoins, Gh and Sp, are known to be substrates for Fpg, Nei, and hNEIL1 BER glycosylases; however, bulky Sp-amine adducts may be more readily repaired by the nucleotide excision repair (NER) pathway. To delineate the contributions of NER and BER for hydantoin lesion repair, a series of Sp-amine adducts of varying size were prepared. In vitro experiments presented herein reveal that UvrABC excision of Gh and Sp is significantly greater than for OG, with rates of Sp removal similar to those observed for known NER substrates. The BER glycosylases Nei, Fpg, and hNEIL1 were also shown to mediate the removal Sp-amine bases from DNA. Detailed kinetic studies performed with hNEIL1 revealed that this glycosylase is relatively insensitive to the size of the Sp-amine adducts and exhibited robust base removal activity under single-turnover conditions for the entire series. Studies suggest that hydantoin lesions may be efficiently repaired in cells by both NER and BER pathways.;Taken together, the work in this dissertation provides unique insight into the chemical features utilized by each of the BER and NER DNA repair enzymes to recognize and mediate repair of oxidatively damaged DNA bases. Illustrating that fine-tuning of overall similar catalytic strategies allows for broad versus specific substrate processing and the exquisite control needed to preserve the genome.
机译：在BER的第一步中，DNA修复糖基化酶从DNA中定位并切除受损的碱基，在基因组的保存和疾病的预防中起着核心作用。大肠杆菌（E. coli）糖基化酶Fpg，MutY和人糖基化酶hOGG1参与了诱变性氧化损伤鸟嘌呤损伤，8-氧代-7,8-二氢鸟嘌呤（8-OxoG或OG）的修复。为了研究用于损伤识别和催化的所需特征，测试了一系列模拟OG特征的取代吲哚核苷酸作为这些糖基化酶的直接底物。结果表明，OG糖基化酶hOGG1尤其是Fpg可以识别和切割非极性OG模拟物，尽管它们缺少氢键基团和碱性原子。与Fpg和hOGG1相比，使用MutY和与A配对的非极性OG类似物进行的单周转动力学实验无法检测到A切除。每种糖基化酶显示的非极性类似物的碱基切除化学的相对速率突出了Fpg，hOGG1和MutY所采用的识别和催化机制的关键差异。DNA修复缺陷涉及多种疾病。这样的例子之一是结肠癌的遗传形式，称为MUTYH相关息肉病（MAP）。 MAP是由MutY（MUTYH）的人类同源物中的缺陷引起的，该缺陷可防止与OG病变相关的G到T转换突变。 MutY是一种独特的糖基化酶，当与OG错配时，可检测并去除正常的腺嘌呤。为了评估MutY和OG之间的相互作用是至关重要的，选择了一系列OG和腺嘌呤核苷酸类似物，它们在碱基对稳定性，氢键能力和糖苷键稳定性方面与天然底物不同。为了确定这些底物修饰影响MutY反应途径的哪一部分，测量了结合亲和力，催化速率和整体细胞修复。本论文报道的结果支持以前的观察结果，表明与OG的任何结构差异均会显着降低结合力，体外有效催化作用以及细菌细胞的成功修复，这表明精确识别OG对于有效MutY活性至关重要。有趣的是，只有在C-2和N-1位置的腺嘌呤修饰对OG：A细胞修复产生负面影响。这表明有效的结合和适度的催化活性不是MutY活性的唯一要求，并且除了在OG：A错配中鉴定OG外，还强调了确认腺嘌呤的重要性。高度诱变的乙内酰脲病灶，胍基乙内酰脲（Gh）和螺亚胺二乙内酰脲（Sp）。有趣的是，在伯胺存在下OG的氧化导致乙内酰脲胺加合物的形成。乙内酰脲Gh和Sp是Fpg，Nei和hNEIL1 BER糖基化酶的底物。但是，大体积的Sp-胺加合物可能更易于通过核苷酸切除修复（NER）途径修复。为了描述NER和BER对乙内酰脲损伤修复的贡献，制备了一系列不同大小的Sp-胺加合物。本文介绍的体外实验表明，Gh和Sp的UvrABC切除显着大于OG，其Sp去除速率与已知NER底物的去除速率相似。 BER糖基化酶Nei，Fpg和hNEIL1也显示出介导从DNA去除Sp-胺碱基的作用。用hNEIL1进行的详细动力学研究表明，该糖基化酶对Sp-胺加合物的大小相对不敏感，并且在整个系列的单周转条件下均表现出强大的碱基去除活性。研究表明，乙内酰脲损伤可通过NER和BER途径在细胞中得到有效修复。总而言之，本论文的研究为BER和NER DNA修复酶各自识别和介导修复所利用的化学特性提供了独特的见解。被氧化破坏的DNA碱基说明总体相似催化策略的微调可实现广泛的与特定的底物处理以及保存基因组所需的精确控制。

著录项

  • 作者

    McKibbin, Paige Lorraine.;

  • 作者单位

    University of California, Davis.;

  • 授予单位 University of California, Davis.;
  • 学科 Chemistry General.;Chemistry Biochemistry.;Chemistry Organic.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 193 p.
  • 总页数 193
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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