Assembly and Activation of Enzyme-ssDNA Complexes

这项研究计划的目的是确定重组酶 在噬菌体中，解旋酶被组装到单链DNA(SsDNA)上 T4DNA复制/重组系统。我们将研究T4 UvsX的组装 重组到突触前细丝，我们将研究两种不同DNA的组装 解旋酶，gp41和dda，在复制分叉和重组中间产物上的单链DNA上。 这三种酶都必须在已经被紧密覆盖的细胞中组装到单链DNA上。 结合Gp32，T4单链DNA结合蛋白。UvsX和gp41都需要 特异性介体蛋白，分别为UvsY或GP59，用于正确组装到Gp32-ssDNA上 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。我们将使用经典生化来探索所有这三种酶的负载机制。 方法(动力学、热力学、荧光法、沉淀法、交联法)、 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突触前的动力学机制 细丝组装和坍塌。我们将测试一个模型，在该模型中，UvsY蛋白选择性地 增强单丝成核，UvsX主动取代单链DNA中的gp32，单丝表现出 与三磷酸腺苷水解有关的动态不稳定性。(2)确定T4GP59蛋白的相互作用 通过复制分叉，DNA控制解旋酶组装和聚合酶阻断。我们将测试一个 Gp32与滞后链单链DNA的协同结合模型将GP59从 聚合酶阻断到解旋酶装载构象，它将gp41解旋酶招募到 复制分叉。(3)确定与Gp32的相互作用如何调节DNA解旋酶 T4Dda蛋白的功能。我们将测试一个模型，在该模型中dda-Gp32蛋白-蛋白质 相互作用促进DDA齐聚并增强其DNA解离性能 复制和重组事务。了解解旋酶和 重组酶正确组装到单链DNA上是理解 DNA复制、重组和修复机制，在所有生物体中都是保守的。 DNA复制/重组/修复机制中的错误与 包括癌症在内的人类疾病状态。了解重组酶和解旋酶是如何 因此，正确组装和激活单链DNA复合体可有助于预防， 癌症的诊断和治疗。

The objective of this research program is to determine the mechanisms by which recombinase and helicase enzymes are assembled onto single-stranded DNA (ssDNA) in the bacteriophage T4 DNA 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 DNA 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 DNA 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 polymerase-blocking 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.