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：
7385078
负责人：
Louis J Romano
金额：
$ 22.59万
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 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7385078
关键词：
2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine 2-aminofluorene Active Sites Adopted Affect Amino Acid Substitution Amino Acids Area Aromatic Amines Aromatic Polycyclic Hydrocarbons Back Benzo(a)pyrene Binding Binding Sites Biochemical Biological Bypass Characteristics Complex DNA DNA Adducts DNA Damage DNA Polymerase I DNA Sequence DNA Structure DNA biosynthesis DNA-Directed DNA Polymerase Duct (organ) structure Experimental Designs Frameshift Mutation Future Goals Grant Hand Helix (Snails)Human Kinetics Lesion Light Measurement Measures Methods Mining Modeling Molecular Molecular Conformation Mutagenesis Mutation N-(deoxyguanosin-8-yl)-2-aminofluorene N-acetyl-2-aminofluorene Nucleotides Oligonucleotides Polymerase Positioning Attribute Property Site Structure Techniques Testing acetylaminofluorene adduct base ear helix experience pol genes

项目摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to gain an understanding of 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. We plan to measure the interactions that occur between the polymerase and these modified templates so that we might understand the molecular mechanism that results in, on the one hand, bypass of a specific lesion (whether error-free or error-prone) or, on the other hand, a blockage of synthesis. Two specific hypotheses will be tested in this current application. The first is that there is a relationship between the conformation of an adduct that is present in DNA and the structure that exists inside the polymerase active site. Second that the adduct induces specific structures to form within the polymerase active site that block the nucleotide binding site, resulting in the inability of a polymerase to undergo a conformational change to the catalytically active ternary complex. Four specific aims are proposed. First, we plan on continuing our studies with site-specifically positioned aromatic amine and polycyclic aromatic hydrocarbon adducts situated in the active site of DNA polymerase I (KF). We will make use of the techniques developed in the prior project period to construct oligonucleotides containing N-acetyl-2-aminofluorene (AAF), 2-aminofluorene (AF) and (+)-trans and (+)-cis-benzo[a]pyrene adducts and extend our models to include 4-amimobiphenyl and PhIP, both of which have structural and mutagenic characteristics that resemble the AF adduct. Second, we will determine how amino acid substitutions within the polymerase active site contribute the properties that effect polymerase mechanism and fidelity. Third, we will use the methods developed in the prior project period to measure the interactions that occur between these carcinogenic adducts and the bypass polymerase, human Pol h (eta). Fourth, we will determine the crystal structures of the T7 DNA polymerase bound to AAF, AF, and B[a]P-modified templates. Taken together, these measurements should help to develop a molecular picture for how these various adducts are accommodated in a polymerase's 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）：本提案的目的是了解分子相互作用，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。我们计划测量聚合酶和这些修饰模板之间发生的相互作用，以便我们了解分子机制，该机制一方面导致绕过特定病变（无论是无错还是易错），或者另一方面另一方面，合成受阻。当前的应用程序将测试两个特定的假设。首先，DNA 中加合物的构象与聚合酶活性位点内部的结构之间存在关系。其次，加合物诱导在聚合酶活性位点内形成特定结构，从而阻断核苷酸结合位点，导致聚合酶无法发生构象变化，形成催化活性三元复合物。提出了四个具体目标。首先，我们计划继续研究位于 DNA 聚合酶 I (KF) 活性位点的特定位点芳香胺和多环芳香烃加合物。我们将利用前期项目期间开发的技术来构建含有N-乙酰基-2-氨基芴（AAF）、2-氨基芴（AF）和（+）-反式和（+）-顺式-苯并[a]的寡核苷酸。 ]芘加合物并将我们的模型扩展为包括 4-氨基联苯和 PhIP，两者都具有类似于 AF 加合物的结构和诱变特征。其次，我们将确定聚合酶活性位点内的氨基酸取代如何影响聚合酶机制和保真度。第三，我们将使用之前项目期间开发的方法来测量这些致癌加合物与旁路聚合酶、人 Pol h (eta) 之间发生的相互作用。第四，我们将确定与 AAF、AF 和 B[a]P 修饰模板结合的 T7 DNA 聚合酶的晶体结构。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。