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首页> 外文学位 >Oxidative damage to DNA: I. Isolation of proteins involved in the recognition of oxidatively damaged DNA. II. Development of next-generation hydroxyl radical footprinting.
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Oxidative damage to DNA: I. Isolation of proteins involved in the recognition of oxidatively damaged DNA. II. Development of next-generation hydroxyl radical footprinting.

机译：对DNA的氧化损伤：I.分离与识别氧化损伤的DNA有关的蛋白质。二。下一代羟基自由基足迹的发展。

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Oxidative damage to DNA has attracted intense research interest with considerable attention focused on base lesions. More than 20 DNA base lesions have been identified, and mechanisms for their repair elucidated. However, for oxidative lesions in the DNA backbone, the nature of the damage-sensing step in their cellular repair remains unclear.;In this dissertation, I examined a synthesized, gapped DNA probe as a model system. The structure of the lesion in the gapped DNA probe corresponds to one type of break found in oxidatively damaged DNA produced by the hydroxyl radical. Electrophoretic Mobility Shift Assay (EMSA) experiments demonstrated an appreciable shift due to the specific binding of yeast proteins to gapped DNA. To isolate the specific binding proteins, I subjected yeast protein extract to fractionation, EMSA gel purification and electroelution, and then electrophoresed the electroeluted proteins on an SDS gel. I then excised the candidate protein bands to prepare samples for mass spectrometry analysis to initially characterize the gapped DNA-binding protein.;The second part of the dissertation presents a new protocol for next-generation hydroxyl radical footprinting that incorporates the Solexa sequencing platform. Emerging next-generation DNA sequencing technologies work up to 200 times faster and cheaper than the conventional Sanger method, and have already brought profound changes to genomic research. In my protocol, I used the Solexa platform, one of the next-generation sequencing technologies, to map hydroxyl radical cleavage patterns in genomic DNA. To meet the Solexa platform's sample requirements, fragmented DNA must fall in a size range of 100-200 bp. Optimization of the conditions of hydroxyl radical cleavage can achieve this objective. Then I used T7 endonuclease I to convert gap sites into double-stranded breaks. Solexa sequencing of DNA samples produced with my protocol agrees well with the theoretically predicted cleavage pattern. The new method will enable researchers to expand the application of hydroxyl radical footprinting to the genome-wide scale with high efficiency.
机译：对DNA的氧化损伤已经引起了广泛的研究兴趣，并将重点放在基础病变上。已经鉴定出20多个DNA碱基病变，并阐明了其修复机制。然而，对于DNA骨架中的氧化损伤，其细胞修复中损伤感知步骤的性质仍不清楚。在本论文中，我研究了一种合成的，缺口的DNA探针作为模型系统。带缝隙的DNA探针中的病变结构对应于由羟基自由基产生的氧化损伤DNA中发现的一种断裂类型。电泳迁移率变动分析（EMSA）实验表明，由于酵母蛋白质与空缺DNA的特异性结合，出现了明显的变动。为了分离特异性结合蛋白，我将酵母蛋白提取物进行了分级分离，EMSA凝胶纯化和电洗脱，然后在SDS凝胶上电泳了电洗脱的蛋白。然后，我切除了候选蛋白条带，以准备用于质谱分析的样品，从而初步鉴定了空缺的DNA结合蛋白。论文的第二部分提出了一种新的用于Solenta测序平台的下一代羟基自由基印迹方法。新兴的下一代DNA测序技术的工作速度比传统的Sanger方法快200倍，而且价格便宜，并且已经为基因组研究带来了深刻的变化。在我的实验方案中，我使用了Solexa平台（下一代测序技术之一）来绘制基因组DNA中的羟基自由基裂解模式。为了满足Solexa平台的样品要求，片段化的DNA大小必须在100-200 bp之间。优化羟基自由基裂解的条件可以达到这个目的。然后，我使用T7核酸内切酶I将缺口位点转换为双链断裂。用我的方案生产的DNA样品的Solexa测序与理论预测的裂解模式非常吻合。新方法将使研究人员能够高效地将羟基自由基足迹的应用扩展到全基因组范围。

著录项

  • 作者

    Xu, Long.;

  • 作者单位

    Boston University.;

  • 授予单位 Boston University.;
  • 学科 Biology Molecular.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 112 p.
  • 总页数 112
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;生物化学;
  • 关键词

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