ASSEMBLY AND ACTIVATION OF ENZYME/SSDNA COMPLEXES

描述(改编自研究人员摘要)：Dr. 莫里奇的研究是为了表征蛋白质--单链DNA和蛋白质--蛋白质 酶复合体组装和激活所需的相互作用 单链DNA。两种不同代表性的酶-单链DNA组装 噬菌体T4 DNA重组和复制系统中存在的问题 将检查：(1)包含T4的突触前细丝的组装 与单链DNA结合的uvsX重组酶；以及(2)必需DNA gp41的组装 噬菌体原粒的解旋酶成分，结合到单链DNA上。一个共同的特点是 这两种酶必须在已经存在的细胞中组装到单链DNA上 被紧密结合的gp32覆盖，T4单链DNA结合蛋白。另一个 共同的特点是，两种酶都需要第二个“组装因子”(uvsY 或GP59)，用于组装到gp32-ssDNA复合体上。这个项目 重点介绍了uvsY用于加载uvsX的详细生化机制， 通过gp59将gp41负载到gp32覆盖的单链DNA分子上。具体的 目标如下：目标1结构和生化特征 UvsY蛋白和uvsY-ssDNA复合体的研究。以下是具体的 将检验假设：单链DNA与六聚体形式的uvsY结合，并 导致uvsY六聚体的显著结构重排； UvsY结合导致符合以下条件的单链DNA碱基的广泛去堆叠 单链DNA的包裹或其他结构扭曲。目标2：描述 UvsY蛋白与gp32-2ssDNA和uvsX-ssDNA复合体的相互作用这个 将测试以下特定假设：uvsY保留其 与gp32和uvsX蛋白的复合体中的六聚体；以及uvsY 结合破坏gp32-ssDNA相互作用的同时稳定uvsX-ssDNA 互动。目的3研究GP59蛋白与蛋白质的相互作用 Gp32-ssDNA复合体。我们将检验以下具体假设： 与gp32-ssDNA结合的关键的gp59分子簇形成了 Gp41解旋酶的最小组装位置；以及gp59破坏稳定 通过蛋白质-蛋白质相互作用与单链DNA结合的gp32-单链DNA复合体 Gp32分子。目的#4研究GP59蛋白与细胞因子的相互作用 Gp41DNA解旋酶。具体假设是GP59促进了 将测试gp41的六角化。

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.