Biological Consequences of Site-specific Damage to DNA

DNA 位点特异性损伤的生物学后果

基本信息

批准号：
8215629
负责人：
Louis J Romano
金额：
$ 23.94万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-05-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8215629
关键词：
2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine 4-biphenylamine Active Sites Address Adopted Affect Aromatic Amines Base Pairing Benzo(a)pyrene Binding Biological Budgets Bypass Carcinogens Cells DNA DNA Adducts DNA Binding DNA Primers DNA Sequence DNA biosynthesis DNA-Directed DNA Polymerase Eukaryotic Cell Fluorescence Resonance Energy Transfer Frameshift Mutation Gel Goals Health Individual Light Malignant Neoplasms Measurement Measures Methods Molecular Molecular Conformation Movement Mutagenesis Mutation Polymerase Positioning Attribute Property Proteins Publishing Research Resolution Rest Site Stereoisomer Structure Surface Plasmon Resonance Techniques Testing Time Work adduct base benzo(a)pyrene-DNA adduct cancer initiation chemical carcinogen design fluorophore innovation interdisciplinary approach novel research study response single molecule

项目摘要

DESCRIPTION (provided by applicant): Gaining an understanding of how a DNA polymerase interacts with the adducts formed by chemical carcinogens is an important goal since these interactions are the basis for many of the adduct-induced effects. The proposed research builds on our prior work that sought to understand the molecular interactions that contribute to the ability of a DNA polymerase to carry out synthesis on a template modified with a bulky carcinogenic adduct. In the current application, we propose to test the central hypothesis that the positioning of an adduct in the polymerase active site is dependent on the adduct structure and DNA sequence context and that specific structures promote specific mechanistic consequences. To accomplish this goal, we have designed a research plan that takes advantage of several novel experimental methods we have developed to measure these interactions between DNA polymerases and several well-defined DNA adducts. First, we will use the intrinsic fluorescent properties of the adducts we have studied in the past to allow us to use them as FRET donors to measure the position of these adducts in the polymerase active site. Second, we will use a single-molecule approach to measure the binding and dynamics of a polymerase during DNA synthesis with single base pair resolution on templates containing bulky DNA adducts in real time. This technique will be applied to both high-fidelity and bypass polymerases and adducts having very different structures in DNA. Third, we will continue to determine the effect of adduct structure and sequence context on polymerase-DNA binding using surface plasmon resonance, a technique that is more sensitive and accurate than the gel-based methods used in the past. Finally, we will continue the crystallographic studies of DNA polymerases bound to templates modified with carcinogenic DNA adducts. Taken together, these measurements should help to develop a molecular picture for how these various adducts are accommodated in the DNA polymerase active site and provide a better understanding of the molecular mechanism of mutagenesis and bypass synthesis that occurs during DNA replication. PUBLIC HEALTH RELEVANCE: In the broadest terms, the relevance of this work rests on the importance of the carcinogenic agents we propose to study in the initiation of cancer in eukaryotic cells. Gaining an understanding of the mutagenic response to the presence of these structures on the cell's DNA is of undeniable importance. More specifically with regards to the need to understand how these carcinogens cause mutations, it is important to study the response of a DNA polymerase to the presence of these adducts, interactions that are known to be dependent on a multitude of factors-most important among these being the adduct structure, the sequence context within which the adduct lies, and the particular structure of the DNA polymerase that must interact with these structures.

描述（由申请人提供）：了解 DNA 聚合酶如何与化学致癌物形成的加合物相互作用是一个重要目标，因为这些相互作用是许多加合物诱导效应的基础。拟议的研究建立在我们之前的工作基础上，该工作旨在了解分子相互作用，这些相互作用有助于 DNA 聚合酶在用大体积致癌加合物修饰的模板上进行合成的能力。在当前的应用中，我们建议测试中心假设，即加合物在聚合酶活性位点中的定位取决于加合物结构和DNA序列背景，并且特定结构促进特定的机械结果。为了实现这一目标，我们设计了一项研究计划，利用我们开发的几种新颖的实验方法来测量 DNA 聚合酶和几种明确的 DNA 加合物之间的相互作用。首先，我们将利用我们过去研究过的加合物的固有荧光特性，使我们能够将它们用作 FRET 供体来测量这些加合物在聚合酶活性位点中的位置。其次，我们将使用单分子方法实时测量 DNA 合成过程中聚合酶的结合和动态，并在包含大量 DNA 加合物的模板上进行单碱基对分辨率。该技术将应用于高保真聚合酶和旁路聚合酶以及 DNA 中具有非常不同结构的加合物。第三，我们将继续使用表面等离子共振确定加合物结构和序列背景对聚合酶-DNA 结合的影响，这种技术比过去使用的基于凝胶的方法更灵敏、更准确。最后，我们将继续对与用致癌 DNA 加合物修饰的模板结合的 DNA 聚合酶进行晶体学研究。总而言之，这些测量结果应有助于绘制一幅关于这些不同加合物如何适应 DNA 聚合酶活性位点的分子图景，并提供对 DNA 复制过程中发生的诱变和旁路合成的分子机制的更好理解。公共卫生相关性：从最广泛的角度来看，这项工作的相关性取决于我们建议研究的致癌物质在真核细胞癌症发生过程中的重要性。了解细胞 DNA 上这些结构的诱变反应具有不可否认的重要性。更具体地说，为了了解这些致癌物如何引起突变，研究 DNA 聚合酶对这些加合物存在的反应非常重要，已知这些相互作用取决于多种因素，其中最重要的是是加合物结构、加合物所在的序列背景以及必须与这些结构相互作用的DNA聚合酶的特定结构。