Error Correction in DNA Synthesis: A Biochemical Study

DNA 合成中的错误纠正：一项生化研究

• 批准号: 8197656
• 负责人: MYRON GOODMAN
• 金额: $ 40.26万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197656
• 关键词: Address; Aging; Amino Acids; Anisotropy; Bacteria; Behavior; Binding; Biochemical; Biological Assay; Catalysis; Cell Death; Cells; Collaborations; Complex; Cytidine Deaminase; DNA; DNA Damage; DNA Replication Damage; DNA biosynthesis; DNA polymerase V; DNA-Directed DNA Polymerase; Disease; Dissociation; Energy Transfer; Ensure; Enzymes; Escherichia coli; Evolution; Exonuclease; Family; Filament; Fluorescent Probes; Frequencies; Gene Silencing; Goals; Grant; HIV-1; Health; Hereditary Disease; Human; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; In Vitro; Individual; Induced Mutation; Inherited; Investigation; Kinetics; Letters; Location; Malignant Neoplasms; Measures; Mediating; Methods; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutate; Mutation; Neurodegenerative Disorders; Nucleoproteins; Organism; Polymerase; Property; Proteins; Regulation; Research; Role; Site; Solutions; Son of Sevenless Proteins; Staging; Techniques; Thinking; Time; UV Mutagenesis; Work; aqueous; base; chromosome replication; crosslink; fitness; functional group; immunological diversity; in vivo; insight; member; monomer; mutant; public health relevance; research study

DESCRIPTION (provided by applicant): An investigation of the biochemical basis of mutation is fundamental to human health related issues involving genetic disorders including cancer, aging, and neurodegenerative disease. An understanding of DNA polymerase fidelity is at the core of understanding how mutations are generated. Our grant, "Error Correction in DNA Synthesis: A Biochemical Study" has, for the past 35 years, focused on fundamental issues of polymerase fidelity. Initially, we developed concepts and techniques to analyze how polymerases select right from wrong bases for insertion into DNA and to eliminate errors through exonuclease proofreading. The scope of our biochemical studies expanded into the field of human immunological diversity, where we studied the properties of DNA-dependent cytidine deaminases involved in the initiation of somatic hypermutation in immunoglobulin genes and inactivation of HIV-1. While investigating the biochemical basis of SOS damaged- induced mutagenesis in E. coli, we discovered DNA polymerase V, a founding member of a new family (Y- family) of "error-prone" DNA polymerases. We showed that pol V is a heterotrimer (UmuD'2C) composed of two proteins required for UV mutagenesis. In 2009, we resolved a long-standing issue in DNA damage-induced mutagenesis in E. coli, the direct role of a RecA nucleoprotein filament (RecA*) in the replication of damaged DNA templates by pol V. We showed that the role of RecA* is to transfer a molecule of RecA7ATP from its 3'- end to convert inactive pol V into mutagenically active pol V Mut. The properties of pol V Mut (UmuD'2C- RecA7ATP) are regulated through a biochemical cycle of polymerase activation, translesion DNA synthesis, deactivation and reactivation. All forms of pol V Mut retain UmuD'2C-RecA7ATP in a bound complex. In this grant, we propose to study each conformational state of pol V Mut by examining where RecA7ATP binds in relation to UmuD'2 and to the catalytic UmuC subunit and observe the transitions between states in real-time. We will incorporate unnatural amino acids in each subunit to attach site-directed fluorescent probes. These probes will be used to investigate each stage of the pol V Mut cycle by stopped-flow FRET and rotational anisotropy techniques. Aim 1 will determine specific interactions between the RecA7ATP, UmuD'2 and UmuC subunits in the activated and deactivated forms of pol V Mut. Aim 2 will investigate individual kinetic steps during polymerase activation, DNA synthesis, deactivation and reactivation. To obtain a deeper understanding of the biochemical properties of pol V Mut in relation to its behavior in the cell, Aim 3 will analyze two "classical" RecA mutants, one that does not induce mutations in the presence of DNA damage and the other which causes hypermutation in the absence of DNA damage. Our proposal addresses a new model for the regulation of DNA damaged-induced mutagenesis, where the active and inactive forms of the DNA polymerase are governed by the assembly of RecA nucleoprotein filament. This new regulatory mechanism acts to ensure that error-prone pol V Mut cannot mutate the cell unnecessarily by copying undamaged DNA templates. PUBLIC HEALTH RELEVANCE: In all organisms including bacteria and humans, mutations are typically deleterious, causing numerous sporadic and inherited diseases. Yet it is clearly evident that mutations are required for evolution and are essential in providing immunological diversity and general fitness. The proposed research explores the biochemical mechanisms of a completely new type of error-prone DNA polymerase, one which is activated when needed to copy damaged DNA, deactivated to keep it from mutating undamaged DNA, then reactivated again to deal with further DNA damage. This study explores biochemical mechanisms that govern the ability of error-prone DNA polymerases to copy damaged DNA that would otherwise cause a cessation of chromosome replication resulting in cell death.

描述（由申请人提供）：对突变的生化基础的研究对于涉及遗传性疾病（包括癌症、衰老和神经退行性疾病）的人类健康相关问题至关重要。了解 DNA 聚合酶保真度是了解突变如何产生的核心。过去 35 年来，我们的资助“DNA 合成中的错误校正：一项生化研究”一直专注于聚合酶保真度的基本问题。最初，我们开发了概念和技术来分析聚合酶如何从错误的碱基中选择正确的碱基插入 DNA，并通过核酸外切酶校对消除错误。我们的生化研究范围扩展到人类免疫多样性领域，我们研究了 DNA 依赖性胞苷脱氨酶的特性，这些酶参与免疫球蛋白基因体细胞超突变的启动和 HIV-1 的失活。在研究大肠杆菌中 SOS 损伤诱导突变的生化基础时，我们发现了 DNA 聚合酶 V，它是“易错”DNA 聚合酶新家族（Y 家族）的创始成员。我们证明 pol V 是一种异源三聚体 (UmuD'2C)，由紫外线诱变所需的两种蛋白质组成。 2009 年，我们解决了大肠杆菌中 DNA 损伤诱导诱变中长期存在的问题，即 RecA 核蛋白丝 (RecA*) 在 pol V 受损 DNA 模板复制中的直接作用。我们表明，RecA* 的作用是将 RecA7ATP 分子从其 3' 端转移，将无活性的 pol V 转化为具有诱变活性的 pol V Mut。 pol V Mut (UmuD'2C-RecA7ATP) 的特性通过聚合酶激活、跨损伤 DNA 合成、失活和再激活的生化循环进行调节。所有形式的 pol V Mut 都将 UmuD'2C-RecA7ATP 保留在结合复合物中。在这项资助中，我们建议通过检查 RecA7ATP 与 UmuD'2 和催化 UmuC 亚基的结合位置来研究 pol V Mut 的每种构象状态，并实时观察状态之间的转变。我们将在每个亚基中掺入非天然氨基酸以附着定点荧光探针。这些探针将用于通过停流 FRET 和旋转各向异性技术研究 pol V Mut 循环的每个阶段。目标 1 将确定 pol V Mut 的激活和失活形式中 RecA7ATP、UmuD'2 和 UmuC 亚基之间的特定相互作用。目标 2 将研究聚合酶激活、DNA 合成、失活和重新激活过程中的各个动力学步骤。为了更深入地了解 pol V Mut 的生化特性及其在细胞中的行为，Aim 3 将分析两种“经典”RecA 突变体，一种在存在 DNA 损伤的情况下不会诱导突变，另一种在不存在 DNA 损伤的情况下引起超突变。我们的提案提出了一种调控 DNA 损伤诱导突变的新模型，其中 DNA 聚合酶的活性和非活性形式由 RecA 核蛋白丝的组装控制。这种新的调控机制可确保容易出错的 pol V Mut 不会通过复制未损坏的 DNA 模板而使细胞发生不必要的突变。 公共卫生相关性：在包括细菌和人类在内的所有生物体中，突变通常是有害的，会导致许多散发性和遗传性疾病。然而，显而易见的是，突变是进化所必需的，并且对于提供免疫多样性和总体适应性至关重要。这项拟议的研究探索了一种全新的易错 DNA 聚合酶的生化机制，这种酶在需要复制受损 DNA 时被激活，失活以防止其突变未受损的 DNA，然后再次重新激活以应对进一步的 DNA 损伤。这项研究探索了控制易错 DNA 聚合酶复制受损 DNA 能力的生化机制，否则会导致染色体复制停止，从而导致细胞死亡。