Assembly and Activation of Enzyme-ssDNA Complexes

描述（由申请人提供）：本研究项目的目的是确定在噬菌体T4 DNA复制/重组系统中重组酶和解旋酶组装到单链DNA （ssDNA）上的机制。我们将研究T4 UvsX重组酶在突触前丝中的组装，我们将研究Gp41和Dda两种不同的DNA解旋酶在复制叉和重组中间体中的组装到ssDNA上。这三种酶必须在已经被紧密结合的Gp32 （T4 ssDNA结合蛋白）覆盖的细胞中组装到ssDNA上。UvsX和Gp41都需要特定的中介蛋白（UvsY或Gp59）的活性才能正确组装到Gp32- ssdna复合物上，而Dda通过与Gp32直接的蛋白-蛋白相互作用来达到相同的效果。我们将使用经典的生化方法（动力学、热力学、荧光、沉降、交联）、单分子方法（荧光成像、力谱）和诱变来探索这三种酶的装载机制。我们的具体目标是：(1)确定UvsX-ssDNA突触前丝组装和崩溃的动力学机制。我们将测试一个模型，其中UvsY蛋白选择性地增强丝的成核，UvsX主动地从ssDNA中取代gp32，并且丝表现出与ATP水解相关的动态不稳定性。(2)确定T4 Gp59蛋白与复制叉DNA的相互作用如何控制解旋酶组装和聚合酶阻断。我们将测试一个模型，其中Gp32与滞后链ssDNA的合作结合将Gp59从聚合酶阻断转化为将Gp41解旋酶招募到复制叉的解旋酶装载构象。(3)确定与Gp32的相互作用如何调节T4 Dda蛋白的DNA解旋酶功能。我们将测试一个模型，其中Dda- gp32蛋白-蛋白质相互作用促进Dda的寡聚化并增强其在复制和重组交易中的DNA解绕特性。了解解旋酶和重组酶如何正确地组装到ssDNA上，对于理解所有生物体中保守的DNA复制、重组和修复机制至关重要。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之间存在明显的联系。因此，了解重组酶和解旋核酸复合物如何正确组装和激活可能有助于癌症的预防、诊断和治疗。

DESCRIPTION (provided by applicant): 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), singlemolecule 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 helicasessDNA complexes are correctly assembled and activated may therefore aid in the prevention, diagnosis, and treatment of cancer. PUBLIC HEALTH REVELANCE: Proper assembly of enzyme-ssDNA complexes is critical for genome replication and maintenance in all organisms. Defects in enzyme-ssDNA assembly processes are implicated in cancer and other human disease states. Enzyme-ssDNA assembly pathways are also potential targets for new classes of antibiotic and antitumor drugs. Our studies of enzyme-ssDNA assembly mechanisms may therefore shed light on how tumors develop and how infectious diseases progress, and may also suggest new treatment strategies.