Assembly and Activation of Enzyme-ssDNA Complexes

描述（由申请人提供）：本研究计划的目的是表征“介体蛋白”在将重组酶和解旋酶组装到单链DNA中的作用。我们的模型是噬菌体14的DNA复制/重组系统。我们将研究含有与ssDNA结合的T4 uvsX重组酶的突触前纤维的组装。我们还将研究组装的gp 41，T4 primosome的必要的DNA解旋酶组分，到ssDNA。这两种酶必须组装到细胞中的ssDNA上，细胞中已经被紧密结合的gp 32（T4 ssDNA结合蛋白）覆盖。在这两种情况下，正确的组装需要特定介体蛋白（分别为uvsY或gp 59）的活性。该项目的重点是uvsY加载uvsX和gp 59加载gp 41到gp 32覆盖的ssDNA分子上的机制。我们的方法是生物化学的，并将利用溶液方法，包括荧光，沉降和亲和层析，加上物理方法，包括X射线晶体学和电子显微镜。我们的具体目标如下：目标#1：研究T4突触前纤维组装的机制。我们将检验以下假设：（A）uvsY六聚体与gp 32-ssDNA的结合破坏了gp 32的协同性，促进了gp 32被uvsX蛋白的置换;（B）uvsY六聚体的结合稳定了uvsX的高亲和力ssDNA结合构象，促进了uvsX-ssDNA丝的成核。目的#2：进行uvsY（T4重组介体蛋白）的X射线晶体学研究。uvsY的原子结构将被单独解析，并与结合的ssDNA和/或gp 32蛋白的衍生物复合。目的#3：研究T4 gp 59蛋白负载解旋酶的机制。我们将检验下列特定的假设：（A）gp 59在gp 32-ssDNA上形成离散的簇，其重塑复合物并促进gp 41解旋酶对gp 32的置换;（B）gp 32的“A-结构域”的结合通过改变gp 59的HMG样“N-结构域”的构象而使gp 59-DNA相互作用不稳定;和（C）gp 59通过诱导或稳定解旋酶的高亲和力NIP结合构象来促进gp 41环六聚体的形成。

DESCRIPTION (provided by applicant): The objective of this research program is to characterize the roles of "mediator proteins" in assembling recombinase and helicase enzymes onto single-stranded DNA. Our model is the DNA replication/recombination system of bacteriophage 14. We will study the assembly of presynaptic filaments containing the T4 uvsX recombinase bound to ssDNA. We will also study the assembly of gp41, the essential DNA helicase component of the T4 primosome, onto ssDNA. Both enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound gp32, the T4 ssDNA-binding protein. In both cases, proper assembly requires the activity of a specific mediator protein (uvsY or gp59, respectively). This project focuses on the mechanisms used by uvsY to load uvsX, and by gp59 to load gp41, onto gp32-covered ssDNA molecules. Our approach is biochemical, and will utilize solution methods including fluorescence, sedimentation, and affinity chromatography, plus physical methods including X-ray crystallography and electron microscopy. Our SPECIFIC AIMS are the following: AIM #1: Investigate the mechanism of T4 presynaptic filament assembly. We will test the following hypotheses: (A) That binding of uvsY hexamers to gp32-ssDNA disrupts gp32 cooperativity, facilitating the displacement of gp32 by uvsX protein: and (B) That binding of uvsY hexamers stabilizes a high-affinity ssDNA-binding conformation of uvsX, facilitating nucleation of uvsX-ssDNA filaments. AIM #2: Perform X-ray crystallography studies of uvsY, the T4 recombination mediator protein. The atomic structure of uvsY will be solved alone and in complex with bound ssDNA and/or derivatives of gp32 protein. AIM #3: Investigate the mechanism of helicase loading by T4 gp59 protein. We will test the following specific hypotheses: (A) That gp59 forms discrete clusters on gp32-ssDNA which remodel the complex and facilitate the displacement of gp32 by gp41 helicase; (B) That binding of gp32's "A-domain" destabilizes gp59-DNA interactions by altering the conformation of gp59's HMG-Iike "N-domain"; and (C) That gp59 promotes gp41 ring-hexamer formation by inducing or stabilizing a high-affinity NIP binding conformation of the helicase.