Mechanisms of Helicases, Translocases and SSB Proteins involved in Genome Maintenance

解旋酶、转位酶和 SSB 蛋白参与基因组维护的机制

• 批准号: 10613926
• 负责人: Timothy M Lohman
• 金额: $ 81.1万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613926
• 关键词: Address; Affect; Allosteric Regulation; Antibiotics; Armadillo Repeat; Bacteria; Binding; Binding Proteins; C-terminal; Communication; Complex; DNA; DNA Binding; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA metabolism; DNA-Binding Proteins; Defect; Development; Disease; Drug Targeting; Escherichia coli; Fluorescence; Genetic Recombination; Genome; Human; Kinetics; Link; Maintenance; Molecular Motors; Motor; Mutation; Organism; Play; Process; Property; Proteins; Role; SS DNA BP; Single-Stranded DNA; Site; Tail; Thermodynamics; Variant; helicase; human DNA; human disease; laser tweezer; melting; novel; nuclease; nucleic acid metabolism; optic tweezer; repaired; single molecule; translocase

Abstract We are studying two classes of DNA binding proteins, DNA helicases and single stranded (ss)DNA binding (SSB) proteins, that are both essential for genome maintenance in all organisms. DNA helicases are ATP-dependent molecular motors that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA replication, recombination and repair. SSB proteins bind tightly to these ssDNA intermediates, protecting the DNA, but also bind directly to at least 17 other SSB interacting proteins (SIPs) to bring them to their sites of action. Defects in DNA helicases are responsible for a number of human diseases. We are undertaking quantitative studies of the mechanisms of DNA unwinding and ssDNA translocation of a multi-subunit DNA helicase/nuclease, E. coli RecBCD, which functions in repair of DNA double strand breaks and recombination, as well as the E. coli UvrD and Rep helicases which function in several DNA repair pathways. RecBCD is a hetero- trimeric complex containing two superfamily 1 (SF1) helicase/translocase motors (RecB, a 3' to 5' motor and RecD, a 5' to 3' motor). Despite extensive study, the mechanism of helicase DNA unwinding is not understood. There is also little known about how the two motors communicate within RecBCD and the allosteric regulation of its motor and nuclease activities by "chi". We have discovered that RecBCD can unwind duplex DNA processively even in the absence of ssDNA translocation by the canonical RecB and RecD motors indicating that DNA melting and ssDNA translocation are separate processes. This ability is regulated by its nuclease and arm domains. We are studying UvrD and Rep to address the question of what is needed to turn a ssDNA translocase into a helicase and how this is regulated. By accessory proteins such as MutL and PriC. Activation of a helicase is not well understood process. E. coli SSB protein is a central player in all aspects of DNA metabolism. It can bind ssDNA in multiple binding modes that differ dramatically in their properties, in particular ssDNA binding cooperativity. A major focus is on the four intrinsically disordered C-terminal tails of SSB that we have shown regulate cooperative binding of SSB to ssDNA and control the binding of the 17 SIPs. We have developed SSB variants that selectively stabilize the different SSB binding modes and that have different numbers of C-terminal tails and different properties and will determine how these affect protein binding and DNA replication. An array of approaches, including thermodynamic, transient kinetic, structural and single molecule approaches (fluorescence and optical tweezers), will be used in these studies.

抽象的 我们正在研究两类的DNA结合蛋白，DNA解旋酶和单链（SS）DNA 结合（SSB）蛋白，这对于所有生物的基因组维持都是必不可少的。脱氧核糖核酸 解旋酶是依赖ATP的分子电机 （SS）DNA复制，重组和修复所需的DNA中间体。 SSB蛋白结合 紧密地与这些ssDNA中间体紧密，保护DNA，但也直接与其他17个SSB结合 相互作用的蛋白质（SIP）将它们带到其作用部位。 DNA解放酶中的缺陷是 负责多种人类疾病。我们正在进行有关 DNA解开的机制和多糖DNA解旋酶/核酸酶的ssDNA易位。 大肠杆菌RECBCD，可在DNA双链断裂和重组的修复中起作用，以及 大肠杆菌UVRD和REP解旋酶在几个DNA修复途径中起作用。 RECBCD是异性恋 包含两个超家族1（SF1）解旋酶/易位酶电动机的三聚体配合物（RECB，3'至5' 电动机和RECD，5'至3'电动机）。尽管进行了广泛的研究，但解旋酶DNA的机制 放松尚不理解。关于这两个电动机如何在其中交流，也鲜为人知 RECBCD及其运动和核酸酶活性的变构调节。我们发现了 即使在没有ssDNA易位的情况下，RECBCD也可以在 规范RECB和RECD电动机，表明DNA熔化和ssDNA易位是独立的 过程。这种能力受其核酸酶和手臂结构域的调节。我们正在研究UVRD和代表 解决将ssDNA易位酶变成解旋酶的问题以及如何将其转化为 受监管。通过辅助蛋白，例如mutl和Pric。解旋酶的激活尚不清楚 过程。大肠杆菌SSB蛋白在DNA代谢的各个方面都是核心参与者。它可以结合ssDNA 多种结合模式在其性质上截然不同，特别是ssDNA结合 合作。主要重点是我们拥有的四个本质上无序无序的C末端尾巴 显示的调节SSB对ssDNA的合作结合并控制17 sip的结合。我们有 开发了SSB变体，可有选择地稳定不同的SSB结合模式，并且具有 不同数量的C末端尾巴和不同的特性，将决定这些如何影响蛋白 结合和DNA复制。一系列方法，包括热力学，瞬态动力学， 结构和单分子方法（荧光和光学镊子）将在这些方法中使用 研究。