Molecular Determinants of DNA Sliding and Hopping by a DNA Repair Glycosylase

DNA 修复糖基化酶 DNA 滑动和跳跃的分子决定因素

• 批准号: 8607469
• 负责人: Meng Meng Rowland
• 金额: $ 5.33万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8607469
• 关键词: Ablation; Affect; Affinity; Base Excision Repairs; Base Pairing; Binding; Binding Sites; Charge; Chemicals; Chimeric Proteins; Cleaved cell; Cytosine; DNA; DNA Binding; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA-Binding Proteins; DNA-Protein Interaction; Deamination; Deoxyribose; Diffusion; Dissection; Electrostatics; Enzymes; Failure; Genome; Genomic DNA; Goals; Human; Human Engineering; Human Genome; Immunoglobulin Somatic Hypermutation; In Vitro; Individual; Island; Kinetics; Knowledge; Lead; Left; Length; Lesion; Ligation; Location; Malignant Neoplasms; Measures; Methods; Microscopic; Molecular; Molecular Probes; Motion; Mutation; Pathology; Pathway interactions; Peptides; Probability; Process; Property; Protein p53; Proteins; Rare Lesion; Role; Site; Slide; Tail; Technology; Testing; Time; Uracil; Variant; Vertebral column; Walking; analog; base; cofactor; design; fascinate; in vivo; inorganic phosphate; methylphosphonate; novel; public health relevance; repair enzyme; repaired; residence; small molecule; tetrahydrofuran; transition mutation; transversion mutation; uracil-DNA glycosylase

DESCRIPTION (provided by applicant): Aberrant uracils in human genomic DNA lead to G:C to A:T transversion mutation, the most frequent mutation found in many cancers. Human uracil DNA glycosylase (hUNG) searches and removes uracil bases from deoxyribose phosphate backbone using a remarkable search mechanism that allows the enzyme to rapidly locate rare lesion sites in a background of roughly 6 billion normal base pairs in the human genome. This general search mechanism has been referred to as "facilitated diffusion", where proteins utilize the DNA chain as a track to accelerate location of their specific sites. Facilitated diffusion uses two microscopic transfer pathways: one-dimensional "sliding" and three-dimensional "hopping". Although numerous studies have established the existence of such a general mechanism, the details of "facilitated diffusion" remain mysterious. The focus of this proposal is to elucidate th molecular interactions that are important for DNA chain tracking by hUNG. Our recently developed "molecular clock" (MC) approach utilizes a small molecule trap to capture transient hopping enzymes while having minimal effect on the sliding ones, thus allowing the dissection of the two tracking pathways individually. Key parameters that can be probed using this approach include the calculation of mean sliding distance, the average distance an enzyme hops away from DNA, and the 1D diffusion constant for DNA sliding. In the first aim, we will use MC approach to test several key aspects of DNA tracking. First, to test the role of DNA phosphate electrostatics, we will insert neutral methylphosphonate linkages in the DNA backbone between two uracil target sites separated by a set number of base pairs, and then measure the effect of charge ablation on the probability that hUNG will slide between the sites. Second, we will test a novel hypothesis that directionally-biased transfer can occur (even in the absence of an energy- providing cofactor) if thermodynamically stable binding sites are inserted between the two uracil target sites. This aim will involve the insertion of spaced high-affinity tetrahydrofuran abasic sie analogues between the uracil sites. The prediction is that a biased walk will occur via high-affinity "island transfer". A biased-walk using basic sites is highly relevant to the in vivo situaion where hUNG must locate and excise clustered uracil sites such as in the process of Ig somatic hypermutation. The second aim is to engineer hUNG-peptide tail variants to have enhanced tracking abilities. Fusion proteins between hUNG and short, positively charged peptides will be constructed using expressed protein ligation technology (EPL) and/or chemical ligation. These peptides will be derived from several DNA binding proteins (p53, HOXD9, H3), and are chosen because they represent various sizes and overall charge, and are known to affect DNA binding and association kinetics. We will measure the fundamental tracking parameters with these variants, which will directly test how the residence time on nonspecific DNA, and the 1D diffusion constant for sliding affect the efficiency of damage repair both in vitro and in vivo.

描述（由申请人提供）：人类基因组DNA中的异常尿嘧啶导致G：C至A：T颠换突变，这是在许多癌症中发现的最常见突变。人尿嘧啶DNA糖基化酶（hUNG）使用一种显着的搜索机制从脱氧核糖磷酸骨架中搜索并去除尿嘧啶碱基，该机制允许该酶在人类基因组中约60亿个正常碱基对的背景中快速定位罕见的病变位点。这种一般的搜索机制被称为“易化扩散”，其中蛋白质利用DNA链作为轨道来加速其特定位点的定位。促进扩散用途 两种微观传递途径：一维“滑动”和三维“跳跃”。尽管许多研究已经确定了这种一般机制的存在，但“促进扩散”的细节仍然是个谜。这个建议的重点是阐明分子间的相互作用是重要的DNA链跟踪hUNG。我们最近开发的“分子钟”（MC）的方法利用小分子陷阱捕捉瞬时跳跃酶，同时对滑动的影响最小，从而允许单独的两个跟踪路径的解剖。使用这种方法可以探测的关键参数包括平均滑动距离的计算，酶从DNA跳离的平均距离，以及DNA滑动的一维扩散常数。在第一个目标中，我们将使用MC方法来测试DNA跟踪的几个关键方面。首先，为了测试DNA磷酸盐静电的作用，我们将在DNA骨架中由一定数量的碱基对分开的两个尿嘧啶靶位点之间插入中性甲基膦酸酯键，然后测量电荷消融对hUNG在位点之间滑动的概率的影响。其次，我们将测试一种新的假设，即如果在两个尿嘧啶靶位点之间插入生物学稳定的结合位点，则可以发生方向性偏向转移（即使在不存在提供能量的辅因子的情况下）。这一目标将涉及在尿嘧啶位点之间插入间隔开的高亲和力四氢呋喃脱碱基类似物。预测是通过高亲和力的“岛转移”将发生偏置行走。使用碱性位点的偏性步移与hUNG必须定位和切除成簇尿嘧啶位点的体内情况高度相关，例如在IG体细胞超突变过程中。第二个目的是工程化hUNG-肽尾变体以具有增强的跟踪能力。hUNG和带正电荷的短肽之间的融合蛋白将使用表达蛋白连接技术（EPL）和/或化学连接来构建。这些肽将衍生自几种DNA结合蛋白（p53、HOXD 9、H3），并且被选择是因为它们代表不同的大小和总电荷，并且已知影响DNA结合和缔合动力学。我们将测量这些变体的基本跟踪参数，这将直接测试在非特异性DNA上的停留时间和滑动的一维扩散常数如何影响体外和体内损伤修复的效率。