Assembly and Activation of Enzyme-ssDNA Complexes

这项研究的目标是确定重组酶和解旋酶的作用机制。 在噬菌体T4DMA中，酶被组装到单链DNA(SsDNA)上 复制/重组系统。我们将研究T4 UvsX重组酶在突触前的组装 细丝，我们将研究两种不同的DMA解旋酶gp41和dda在单链DNA上的组装 复制分叉和重组中间产物。这三种酶都必须装配在单链DNA上。 已经被紧密结合的Gp32(T4单链DNA结合蛋白)覆盖的细胞。UvsX和gp41都是 需要特定的中介蛋白的活性，分别是UvsY或GP59，才能正确地组装到 Gp32-ssDNA复合体，而DDA通过蛋白质-蛋白质直接相互作用达到同样的效果 使用Gp32。我们将使用经典的生物化学方法探索所有这三种酶的负载机制。 (动力学、热力学、荧光、沉淀法、交联法)、单分子方法 (荧光成像、力光谱)和诱变。我们的具体目标是：(1)确定 UvsX-ssDNA突触前纤维组装和崩解的动力学机制。我们将在这里测试一个模型 其中UvsY蛋白选择性地促进细丝成核，UvsX主动取代单链DNA中的gp32， 而细丝表现出与ATP水解有关的动态不稳定性。(2)确定T4的相互作用 GP59蛋白与复制叉DNA控制解旋酶的组装和聚合酶的阻断。我们将测试一个 Gp32与滞后链单链DNA协同结合将GP59从聚合酶阻断转化为Gp59的模型 到将gp41解旋酶招募到复制分叉上的解旋酶负载构象。(三)确定 与Gp32的相互作用如何调节T4Dda蛋白的DNA解旋酶功能。我们将在这里测试一个模型 哪种dda-Gp32蛋白-蛋白质相互作用促进dda寡聚并增强其DNA 解除复制和重组事务中的属性。了解解旋酶和 重组酶在单链DNA上的正确组装是理解DNA的基础 在所有生物体中都保守的复制、重组和修复机制。有明确的 DNA复制/重组/修复机制中的错误与人类疾病状态之间的联系 包括癌症。了解重组酶和解旋酶-单链DNA复合体是如何正确的 因此，组装和激活可能有助于癌症的预防、诊断和治疗。

The objective of this research program is to determine the mechanisms by which recombinase and helicase enzymes are assembled onto single-stranded DMA (ssDNA) in the bacteriophage T4 DMA replication/recombination system. We will study the assembly of the T4 UvsX recombinase into presynaptic filaments, and we will study the assembly of two different DMA helicases, Gp41 and Dda, onto ssDNA at replication forks and in recombination intermediates. All three enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound Gp32, the T4 ssDNA-binding protein. UvsX and Gp41 both require the activity of a specific mediator protein, UvsY or Gp59, respectively, for proper assembly onto Gp32-ssDNA complexes, whereas Dda achieves the same effect through direct protein-protein interactions with Gp32. We will explore all three enzyme loading mechanisms using classical biochemical methods (kinetics, thermodynamics, fluorescence, sedimentation, crosslinking), single-molecule approaches (fluorescence imaging, force spectroscopy), and mutagenesis. Our SPECIFIC AIMS are: (1) Determine the kinetic mechanism of UvsX-ssDNA presynaptic filament assembly and collapse. We will test a model in which UvsY protein selectively enhances filament nucleation, UvsX actively displaces gp32 from ssDNA, and filaments exhibit dynamic instability linked to ATP hydrolysis. (2) Determine how interactions of T4 Gp59 protein with replication fork DMA control helicase assembly and polymerase blockage. We will test a model in which cooperative binding of Gp32 to lagging-strand ssDNA converts Gp59 from a polymeraseblocking to a helicase-loading conformation that recruits Gp41 helicase to the replication fork. (3) Determine how interactions with Gp32 modulate the DNA helicase functions of T4 Dda protein. We will test a model in which Dda-Gp32 protein-protein interactions promote the oligomerization of Dda and enhance its DNA unwinding properties in both replication and recombination transactions. Understanding how helicase and recombinase enzymes are correctly assembled onto ssDNA is fundamental to understanding DNA replication, recombination, and repair mechanisms that are conserved in all organisms. There are clear links between errors in DNA replication/recombination/repair machineries and human disease states including cancer. Understanding how recombinase- and helicase-ssDNA complexes are correctly assembled and activated may therefore aid in the prevention, diagnosis, and treatment of cancer.