Assembly and Activation of Enzyme-ssDNA Complexes

酶-ssDNA复合物的组装和激活

• 批准号: 6544460
• 负责人: SCOTT W MORRICAL
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6544460
• 关键词: DNA binding protein; DNA repair; DNA replication; X ray crystallography; affinity chromatography; bacteriophage T4; electron microscopy; enzyme complex; enzyme induction /repression; fluorescence resonance energy transfer; genetic recombination; helicase; high performance liquid chromatography; intermolecular interaction; protein biosynthesis; protein protein interaction; protein structure function; recombinase; stoichiometry; virus protein

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.

描述（由申请人提供）：该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上的作用。我们的模型是噬菌体14的DNA复制/重组系统。我们将研究包含与ssDNA结合的T4 UVSX重组酶的突触前细丝的组装。我们还将研究T4原始体的必不可少的DNA解旋酶成分GP41的组装到ssDNA上。两种酶必须在细胞中的ssDNA上组装，该细胞已经被紧密结合的GP32（T4 ssDNA结合蛋白）组装。在这两种情况下，适当的组装都需要特定介体蛋白（分别为UVSY或GP59）的活性。该项目的重点是UVSY用于加载UVSX的机制，以及将GP59加载到GP32覆盖的SSDNA分子上。我们的方法是生化的，将利用溶液方法，包括荧光，沉积和亲和色谱法，以及包括X射线晶体学和电子显微镜在内的物理方法。我们的具体目的是：目标＃1：研究T4突触前细丝组件的机制。我们将测试以下假设：（a）UVSY六聚体与GP32-SSDNA的结合破坏GP32的合作性，促进了UVSX蛋白质的GP32的位移，而UVSX蛋白质的位移：（b）uvsy六聚体稳定的uvsy hexamers稳定构成了高度ssdna结合的uvsxxxxxxxxxxxxxxxx，uvsxxx的构造。 UVSX-SSDNA丝。目标＃2：对T4重组介质蛋白进行UVSY进行X射线晶体学研究。 UVSY的原子结构将单独解决，并与结合的ssDNA和/或GP32蛋白的衍生物求解。 AIM＃3：研究T4 GP59蛋白的解旋酶负荷的机理。我们将测试以下特定假设：（a）GP59在GP32-SSDNA上形成离散簇，该簇重塑了复合物并促进了GP41旋转酶的GP32的位移； （b）GP32的“ A-domain”结合通过改变GP59的HMG-IK-IK-IK-IK-IKE“ N-domain”的构象来破坏GP59-DNA相互作用； （c）GP59通过诱导或稳定旋转酶的高亲和力NIP结合构象来促进GP41环形己形成。