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: 10571587
• 负责人: Timothy M Lohman
• 金额: $ 3.32万
• 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/10571587
• 关键词: Address; Affect; Allosteric Regulation; Armadillo Repeat; Binding; Binding Proteins; C-terminal; Complex; DNA; DNA Binding; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA metabolism; DNA-Binding Proteins; Defect; Disease; Escherichia coli; Fluorescence; Genetic Recombination; Genome; Kinetics; Maintenance; Molecular Motors; Motor; Organism; Process; Property; Proteins; SS DNA BP; Single-Stranded DNA; Site; Tail; Thermodynamics; Variant; helicase; human disease; laser tweezer; melting; nuclease; recombinational repair; 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 DNA 结合蛋白：DNA 解旋酶和单链 (ss)DNA 结合（SSB）蛋白，这对于所有生物体的基因组维护都是必需的。脱氧核糖核酸 解旋酶是 ATP 依赖性分子马达，可解旋双链 DNA 形成单链 (ss) DNA 复制、重组和修复所需的 DNA 中间体。 SSB 蛋白结合 与这些 ssDNA 中间体紧密结合，保护 DNA，同时也直接与至少 17 个其他 SSB 结合 相互作用蛋白（SIP）将它们带到作用位点。 DNA 解旋酶的缺陷是 是许多人类疾病的罪魁祸首。我们正在进行定量研究 多亚基 DNA 解旋酶/核酸酶的 DNA 解旋和 ssDNA 易位机制，E. 大肠杆菌 RecBCD，其功能是修复 DNA 双链断裂和重组，以及 大肠杆菌 UvrD 和 Rep 解旋酶在多种 DNA 修复途径中发挥作用。 RecBCD 是一种异质 含有两个超家族 1 (SF1) 解旋酶/转位酶马达的三聚体复合物（RecB，一个 3' 至 5' 电机和 RecD（5' 至 3' 电机）。尽管进行了大量研究，但 DNA 解旋酶的机制 放松不被理解。关于两个电机如何在内部进行通信也知之甚少。 RecBCD 及其运动和核酸酶活性“chi”的变构调节。我们发现 即使在没有 ssDNA 易位的情况下，RecBCD 也可以逐步解开双链 DNA 规范的 RecB 和 RecD 电机表明 DNA 熔解和 ssDNA 易位是分开的 流程。这种能力由其核酸酶和臂结构域调节。我们正在研究 UvrD 和 Rep 解决将 ssDNA 转位酶转变为解旋酶所需的条件以及如何实现这一问题的问题 受监管。由 MutL 和 PriC 等辅助蛋白组成。解旋酶的激活尚不清楚 过程。大肠杆菌 SSB 蛋白在 DNA 代谢的各个方面发挥着核心作用。它可以结合ssDNA 多种结合模式，其特性差异很大，特别是 ssDNA 结合 合作性。主要关注点是我们所拥有的 SSB 的四个本质上无序的 C 末端尾部 显示调节 SSB 与 ssDNA 的协同结合并控制 17 个 SIP 的结合。我们有 开发了 SSB 变体，可以选择性地稳定不同的 SSB 结合模式，并且具有 不同数量的 C 末端尾部和不同的特性将决定它们如何影响蛋白质 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（荧光和光镊）将用于这些 研究。