Error Correction in DNA Synthesis: A Biochemical Study

描述（由申请人提供）：突变的生化基础研究对于涉及遗传疾病（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。对DNA聚合酶保真度的理解是理解突变如何产生的核心。在过去的35年里，我们的资助项目“DNA合成中的纠错：一项生化研究”一直专注于聚合酶保真度的基本问题。最初，我们开发了一些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来消除错误。我们的生物化学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性质，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。在探讨SOS损伤诱发E.在大肠杆菌中，我们发现了DNA聚合酶V，它是一个新的“易错”DNA聚合酶家族（Y家族）的创始成员。我们发现pol V是由紫外诱变所需的两个蛋白质组成的异源三聚体（UmuD'2C）。在2009年，我们解决了一个长期存在的问题，在DNA损伤诱导突变的E。我们发现RecA* 的作用是从它的3 '端转移一个RecA 7ATP分子，将无活性的pol V转化成有诱变活性的pol V Mut。pol V Mut（UmuD'2C-RecA' 7ATP）的性质通过聚合酶活化、跨损伤DNA合成、失活和再活化的生物化学循环来调节。所有形式的pol V Mut都将UmuD ′ 2C-RecA ′ 7ATP保留在结合的复合物中。在这项研究中，我们建议通过检查RecA 7ATP与UmuD '2和催化UmuC亚基的结合来研究pol V Mut的每个构象状态，并实时观察状态之间的转换。我们将在每个亚基中掺入非天然氨基酸以连接定点荧光探针。这些探针将被用来调查的pol V Mut周期的每个阶段，通过停流FRET和旋转各向异性技术。目的1将确定在pol V Mut的活化和失活形式中RecA 7ATP、UmuD '2和UmuC亚基之间的特异性相互作用。目的2将研究聚合酶活化、DNA合成、失活和再活化过程中的各个动力学步骤。为了更深入地了解pol V Mut的生化特性与其在细胞中的行为之间的关系，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。我们的建议解决了一个新的模式，为调节DNA损伤诱导的诱变，其中的活性和非活性形式的DNA聚合酶的RecA核蛋白丝的组装。这种新的调节机制可以确保易错的pol V Mut不能通过复制未受损的DNA模板来不必要地突变细胞。 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，导致许多散发性和遗传性疾病。然而，很明显，突变是进化所必需的，并且在提供免疫多样性和一般适应性方面是必不可少的。这项研究探索了一种全新的易错DNA聚合酶的生化机制，这种聚合酶在需要复制受损DNA时被激活，为了防止未受损DNA发生突变而被灭活，然后再次被激活以处理进一步的DNA损伤。这项研究探讨了控制易错DNA聚合酶复制受损DNA的能力的生化机制，否则会导致染色体复制停止，导致细胞死亡。

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.