Biological Consequences of Site-Specific Damage to DNA

DNA 位点特异性损伤的生物学后果

• 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
• 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

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）：该提案的目的是了解有助于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。我们计划测量聚合酶与这些修饰的模板之间发生的相互作用，以便我们可以理解一方面导致特定病变（无论是无错误还是容易出错）的分子机制，或者另一方面是合成的阻塞。在此当前应用中将测试两个特定的假设。首先是，在DNA中存在的加合物的构象与聚合酶活性位点内的结构之间存在关系。其次，加合物会诱导特定的结构在聚合酶活性位点内形成，从而阻断核苷酸结合位点，从而导致聚合酶无法对催化活性三元复合物进行构象变化。提出了四个具体目标。首先，我们计划继续使用位于DNA聚合酶I（KF）活性位点的位置特定定位的定位芳香胺和多环芳芳烃加合物。 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 AF加合物。其次，我们将确定聚合酶活性位点中的氨基酸取代如何影响聚合酶机制和保真度的特性。第三，我们将使用前一个项目期间开发的方法来衡量这些致癌性加合物与旁路聚合酶人类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。综上所述，这些测量值应有助于开发分子图片，以使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。