Assembly and Activation of Enzyme-ssDNA Complexes

描述(由申请人提供)：本研究计划的目标是确定“中介蛋白”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。我们将研究含有与单链DNA结合的T4 uvsX重组酶的突触前细丝的组装。我们还将研究gp41，T4原始体的基本DNA解旋酶成分，组装到单链DNA上。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，正确的组装都需要特定的中介蛋白(分别是uvsY或GP59)的活性。这个项目的重点是uvsY将uvsX加载到gp32覆盖的单链DNA分子上，以及gp59将gp41加载到gp32覆盖的ssDNA分子上所使用的机制。我们的方法是生化的，将利用包括荧光、沉淀和亲和层析在内的溶液方法，以及包括X射线结晶学和电子显微镜在内的物理方法。我们的具体目标如下：目的1：研究T4突触前微丝组装的机制。我们将检验以下假设：(A)uvsY六聚体与gp32-ssDNA的结合破坏了gp32的协同作用，促进了gp32被uvsX蛋白置换；(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细丝的成核。目的#2：对T4重组介体蛋白uvsY进行X射线结晶学研究。UvsY的原子结构将被单独或与结合的单链DNA和/或gp32蛋白的衍生物组成的复合体解决。目的#3：探讨T4GP59蛋白负载解旋酶的机制。我们将检验以下具体假设：(A)GP59在gp32-ssDNA上形成离散的簇，从而重塑复合体，并促进gp32被gp41解旋酶置换；(B)gp32‘S“A-结构域”的结合通过改变GP59’S HMG-iike“N-结构域”的构象来破坏GP59与DNA的相互作用；以及(C)GP59通过诱导或稳定解旋酶的高亲和力NIP结合构象而促进gp41环-六角体的形成。

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.