ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES

酶-SSDNA 复合物的组装和激活

• 批准号: 2691547
• 负责人: SCOTT W MORRICAL
• 金额: $ 23.08万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2691547
• 关键词: DNA binding protein; DNA replication; bacteriophage T4; circular dichroism; crosslink; enzyme complex; enzyme induction /repression; fluorescence spectrometry; genetic recombination; helicase; high performance liquid chromatography; intermolecular interaction; protein biosynthesis; protein structure function; recombinase; sedimentation; stoichiometry; ultraviolet radiation; virus protein

DESCRIPTION (adapted from investigator's abstract): The objective of Dr. Morrical's research is to characterize the protein-ssDNA and protein-protein interactions required for the assembly and activation of enzyme complexes on single-stranded DNA. Two different representative enzyme-ssDNA assembly problems in the bacteriophage T4 DNA recombination and replication systems will be examined: (1) Assembly of presynaptic filaments containing the T4 uvsX recombinase bound to ssDNA; and (2) Assembly of gp41, the essential DNA helicase component of the phage primosome, onto ssDNA. A common feature is that both enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound gp32, the T4 ssDNA-binding protein. Another common feature is that both enzymes require a second "assembly factor" (uvsY or gp59, respectively) for assembly onto gp32-ssDNA complexes. This project focuses on the detailed biochemical mechanisms used by uvsY to load uvsX, and by gp59 to load gp41, onto gp32-covered ssDNA molecules. The SPECIFIC AIMS are the following: AIM 1 Characterize structural and biochemical aspects of uvsY protein and uvsY-ssDNA complexes. The following specific hypotheses will be tested: that ssDNA binds to a hexameric form of uvsY and causes a significant structural rearrangement of the uvsY hexamer; and that uvsY binding causes extensive unstacking of ssDNA bases consistent with wrapping or other structural distortions of the ssDNA. AIM 2 Characterize interactions of uvsY protein with gp32-2ssDNA and uvsX-ssDNA complexes. The following specific hypotheses will be tested: that uvsY retains its hexameric form in complexes with gp32 and uvsX proteins; and that uvsY binding destabilizes gp32-ssDNA interactions while stabilizing uvsX-ssDNA interactions. AIM 3 Characterize interactions of gp59 protein with gp32-ssDNA complexes. The following specific hypotheses will be tested: that a critical cluster of gp59 molecules bound to gp32-ssDNA forms the minimal assembly site for the gp41 helicase; and that gp59 destabilizes gp32-ssDNA complexes through protein-protein interactions with ssDNA-bound gp32 molecules. AIM # 4 Characterize interactions of gp59 protein with the gp41 DNA helicase. The specific hypothesis that gp59 promotes the hexamerization of gp41 will be tested.

描述（根据调查员的摘要进行了改编）：博士的目的。 Morrical的研究是表征蛋白质-SSDNA和蛋白质 - 蛋白质 组装和激活酶复合物上所需的相互作用 单链DNA。 两个不同的代表性酶 - ssDNA组件 噬菌体T4 DNA重组和复制系统中的问题 将检查：（1）包含T4的突触前细丝组装 与ssDNA结合的UVSX重组酶； （2）GP41的组装，必需的DNA 噬菌体原始体的解旋酶成分，到ssDNA。 一个共同的功能是 这两种酶必须在已经已经存在的细胞中的ssDNA上 T4 ssDNA结合蛋白被紧密结合的GP32覆盖。 其他 共同特征是这两个酶都需要第二个“装配因子”（UVSY 或GP59分别用于组装到GP32-SSDNA复合物上。 这个项目 专注于UVSY使用的详细生化机制，用于加载UVSX， 并由GP59加载GP41，将其加载到GP32覆盖的ssDNA分子上。 具体 目的是以下内容：AIM 1表征结构和生化 UVSY蛋白和UVSY-SSDNA复合物的各个方面。 以下特定 将测试假设：ssDNA与uvsy的六聚体形式结合 引起UVSY六聚体的显着结构重排；那 UVSY结合导致ssDNA碱基的广泛拆卸与 包裹或其他结构扭曲的ssDNA。 目标2特征 UVSY蛋白与GP32-2SSDNA和UVSX-SSDNA复合物的相互作用。 这 以下特定假设将进行测试：UVSY保留其 与GP32和UVSX蛋白的复合物中的六聚体形式；那uvsy 结合稳定UVSX-SSDNA时，破坏了GP32-SSDNA相互作用 互动。 AIM 3表征GP59蛋白与 GP32-SSDNA复合物。 将测试以下特定假设： 与GP32-SSDNA结合的临界GP59分子簇形成了 GP41解旋酶的最小装配位点； GP59破坏了 通过蛋白质 - 蛋白质相互作用与ssDNA结合的GP32-SSDNA复合物 GP32分子。 AIM＃4表征GP59蛋白与 GP41 DNA解旋酶。 GP59促进的具体假设 将测试GP41的六聚体。