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：
8616030
负责人：
Louis J Romano
金额：
$ 23.22万
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 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8616030
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 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 public health relevance 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.

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加合物引起的许多效应的基础。拟议的研究基于我们先前的工作，该研究试图了解有助于DNA聚合酶在用笨重的致癌加合物修饰的模板上进行合成的能力的分子相互作用。在当前的应用中，我们建议测试中心假设，即在聚合酶活性位点中的加合物的定位取决于加合物结构和DNA序列上下文，并且特定结构促进了特定的机械后果。为了实现这一目标，我们设计了一个研究计划，该计划利用了我们开发的几种新型实验方法，以测量DNA聚合酶和几个定义明确的DNA加合物之间的这些相互作用。首先，我们将使用过去研究的加合物的固有荧光性能，以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，我们将使用一种单分子方法来测量DNA合成过程中聚合酶的结合和动力学，并在含有大量DNA加合物的模板上实时分辨率分辨率实时。该技术将应用于具有截然不同的DNA结构的高保真和旁路聚合酶和加合物。第三，我们将继续确定使用表面等离子体共振的加合物结构和序列上下文对聚合酶-DNA结合的影响，这种技术比过去使用的基于凝胶的方法更敏感和准确。最后，我们将继续对与致癌DNA加合物修饰的模板结合的DNA聚合酶的晶体学研究。综上所述，这些测量值应有助于开发分子图片，以在DNA聚合酶活性位点如何容纳这些各种加合物，并更好地理解DNA复制过程中发生的诱变和旁路合成的分子机制。