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

摘要 我们正在研究两类DNA结合蛋白，DNA解旋酶和单链（ss）DNA 结合蛋白（SSB），这两个都是在所有生物体的基因组维护必不可少的。DNA 解旋酶是ATP依赖性分子马达，其将双链DNA解旋以形成单链 (ss)DNA复制、重组和修复所需的DNA中间体。SSB蛋白结合 紧密结合这些ssDNA中间体，保护DNA，但也直接结合至少17个其他SSB 相互作用蛋白（SIP）将它们带到它们的作用位点。DNA解旋酶的缺陷是 导致了许多人类疾病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转位机制，E. 大肠杆菌RecBCD，其功能是修复DNA双链断裂和重组，以及 E. coliUvrD和Rep解旋酶在几种DNA修复途径中起作用。RecBCD是一个异性恋- 含有两个超家族1（SF 1）解旋酶/转位酶马达（RecB，3'至5'端的一种三聚体复合物， 马达和RecD，5'到3'马达）。尽管有广泛的研究，解旋酶DNA的机制， 解旋不被理解。关于这两个马达内部是如何交流的也知之甚少 RecBCD及其马达和核酸酶活性的“chi”变构调节。我们已经发现 RecBCD即使在没有ssDNA易位的情况下，也可以解旋双链DNA原丝， 典型的RecB和RecD马达表明DNA解链和ssDNA易位是分开的 流程.这种能力受其核酸酶和臂结构域的调节。我们正在研究UvrD和Rep， 解决的问题是什么需要把ssDNA易位酶成解旋酶，这是如何 监管.辅助蛋白如MutL和PriC。解旋酶的激活还不是很清楚 过程E. coli SSB蛋白在DNA代谢的各个方面都起着重要作用。它可以结合ssDNA 多种结合模式，其性质差异很大，特别是ssDNA结合 协同性一个主要的焦点是在四个本质上无序的C-末端尾部的SSB，我们有 显示调节SSB与ssDNA的协同结合并控制17个SIP的结合。我们有 开发了选择性稳定不同SSB结合模式的SSB变体， 不同数量的C末端尾和不同的性质，并将决定这些如何影响蛋白质 结合和DNA复制。一系列的方法，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），将用于这些 问题研究

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.