Assembly and Activation of Enzyme-ssDNA Complexes

描述（由申请人提供）：本研究项目的目的是描述“中介蛋白”在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。我们将研究包含T4 uvsX重组酶与ssDNA结合的突触前细丝的组装。我们还将研究gp41在ssDNA上的组装，gp41是T4原体必不可少的DNA解旋酶成分。这两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。在这两种情况下，适当的组装都需要特定中介蛋白（分别为uvsY或gp59）的活性。本项目重点研究了uvsY将uvsX和gp59将gp41加载到gp32覆盖的ssDNA分子上的机制。我们的方法是生化，并将利用溶液方法，包括荧光，沉淀，亲和层析，加上物理方法，包括x射线晶体学和电子显微镜。我们的具体目的如下：目的1：研究T4突触前丝组装的机制。我们将测试以下假设：(A) uvsY六聚体与gp32- ssdna的结合破坏了gp32的协同性，促进了gp32被uvsX蛋白取代；(B) uvsY六聚体的结合稳定了uvsX高亲和力的ssdna结合构象，促进了uvsX- ssdna细丝的成核。目的2：对T4重组中介蛋白uvsY进行x射线晶体学研究。uvsY的原子结构将单独或与结合的ssDNA和/或gp32蛋白衍生物一起解决。目的3：研究T4 gp59蛋白装载解旋酶的机制。我们将测试以下具体假设：(A) gp59在gp32- ssdna上形成离散簇，从而重塑复合物并促进gp41解旋酶取代gp32；(B) gp32“a结构域”的结合通过改变gp59的hmg样“n结构域”的构象来破坏gp59- dna相互作用的稳定性；(C) gp59通过诱导或稳定解旋酶的高亲和力NIP结合构象来促进gp41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.