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（改编自研究者摘要）：Dr. 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所需的相互作用 单链DNA 两种不同的代表性酶-ssDNA组装 噬菌体T4 DNA重组和复制系统中的问题 将检查：（1）含有T4的突触前细丝的组装 uvsX重组酶与ssDNA的结合;（2）gp 41的组装， 噬菌体启动体的解旋酶组分，到ssDNA上。 一个共同的特点是 这两种酶必须组装到细胞中的ssDNA上， 覆盖有紧密结合的gp 32，T4 ssDNA结合蛋白。 另一 两种酶的共同特征是都需要第二个“组装因子”（uvsY 或gp 59）组装到gp 32-ssDNA复合物上。 这个项目 侧重于详细的生化机制所使用的uvsY加载uvsX， 并通过gp 59将gp 41加载到gp 32覆盖的ssDNA分子上。 具体 目的如下：目的1表征结构和生物化学 uvsY蛋白和uvsY-ssDNA复合物的方面。 以下具体 假设将被测试：ssDNA结合六聚体形式的uvsY， 引起uvsY六聚体的显著结构重排;并且 uvsY结合导致ssDNA碱基的大量解堆积， ssDNA的缠绕或其他结构扭曲。 AIM 2表征 uvsY蛋白与gp 32 -2ssDNA和uvsX-ssDNA复合物的相互作用。 的 将测试以下特定假设：uvsY保留其 与gp 32和uvsX蛋白复合六聚体形式;和uvsY 结合使gp 32-ssDNA相互作用不稳定，同时使uvsX-ssDNA稳定 交互. 目的3研究gp 59蛋白与 gp 32-ssDNA复合物。 将检验以下特定假设： 与gp 32-ssDNA结合的gp 59分子的关键簇形成了 gp 41解旋酶的最小装配位点; gp 59使 通过与ssDNA结合的蛋白质-蛋白质相互作用的gp 32-ssDNA复合物 gp 32分子。 目的#4表征gp 59蛋白与 gp 41 DNA解旋酶。 关于gp 59促进 将测试GP 41的六聚化。