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.

摘要 我们正在研究两类dna结合蛋白，dna解旋酶和单链dna。 结合蛋白(SSB)，对所有生物体的基因组维持都是必不可少的。脱氧核糖核酸 解旋酶是依赖于ATP的分子马达，可以解开双链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 紧密地与这些单链DNA中间体结合，保护DNA，但也直接与至少17个其他单链B结合 相互作用蛋白(Sip)将它们带到它们的作用部位。DNA解旋酶的缺陷有 对人类的许多疾病负有责任。我们正在进行定量研究 多亚单位DNA解旋酶/核酸酶的DNA解离和单链DNA易位的机制。 ColiRecBCD，它在DNA双链断裂和重组修复中起作用，以及 E.ColiUvrD和Rep解旋酶，它们在几个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(RecB，3‘至5’)的三聚体复合体 马达和RecD，5‘到3’马达)。尽管有广泛的研究，解旋酶DNA的机制 解体是不被理解的。关于这两个马达是如何在体内交流的也鲜为人知 RecBCD及其“气”对其马达和核酸酶活性的变构调节。我们发现 即使在没有单链DNA易位的情况下，RecBCD也可以连续地解开双链DNA 典型的RecB和RecD马达表明DNA融化和单链DNA易位是分开的 流程。这种能力受其核酸酶和ARM结构域的调节。我们正在研究Uvrd和Rep to 解决需要什么才能将单链DNA转位酶转变为解旋酶的问题以及这是如何实现的 受监管的。由MutL、PrIC等辅助蛋白调控。解旋酶的激活还不是很清楚 进程。大肠杆菌SSB蛋白在DNA新陈代谢的各个方面都起着核心作用。它能与单链DNA结合 多种结合模式，它们的性质有很大不同，特别是单链DNA结合 协作性。一个主要的焦点是我们拥有的SSB的四个内在无序的C末端尾巴 显示调节SSB与单链DNA的协同结合，并控制17个sip的结合。我们有 开发了选择性稳定不同SSB结合模式的SSB变体，并具有 不同数量的C末端尾巴和不同的性质，将决定这些如何影响蛋白质 结合和DNA复制。一系列方法，包括热力学、瞬变动力学、 结构和单分子方法(荧光和光学镊子)将用于这些 学习。