ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES

这项研究计划的主要目标是阐明分子 单链DNA上酶复合体的组装机制 并被激活。特定的酶与单链DNA的相互作用是一种 DNA复制机制的基本特征。重组，以及 发生在所有活细胞中的修复。细胞的组装和激活 然而，由于螺旋的存在，酶-单链DNA复合体是复杂的。 紧密结合并协同作用的不稳定蛋白(HDP) 单链DNA。为了与单链DNA结合并激活其催化活性，一种酶 必须首先取代高密度脂蛋白，这是一个对能源不利的过程。因此 似乎需要特殊的HDP位移机构来加载 酶作用于预先存在的HDP-单链DNA基质上。我们建议对这些问题进行研究 通过研究分子台阶的位移和加载机制 参与了两种不同模型酶的组装和激活- 单链DNA复合体：噬菌体T4基因41的DNA解旋酶 蛋白(Gp41)和通用重组酶T4uvsX蛋白。 每种酶-单链DNA复合体的组装和激活似乎 需要大量的蛋白质-单链DNA和/或蛋白质-蛋白质相互作用 作为三种不同的噬菌体蛋白：酶本身，一种酶特有的 装配因子，以及一种特异的HDP--T4基因32蛋白(Gp32)。我们的 因此，特定的目标是剖析蛋白质-单链DNA和蛋白质- 参与gp41和uvsX负载到gp32上的蛋白质相互作用- 单链DNA。这将通过分析两者的交互作用来实现 组装因子与单链DNA、gp32和它们的酶配对。 蛋白质-单链DNA的相互作用将直接用荧光法进行检测 光谱分析和偶合分光光度间接测定。蛋白质- 蛋白质的相互作用将通过蛋白质亲和力的组合来检验。 层析和沉淀法，并通过分离和 特定改变的蛋白质的特征。预计 这些研究将确定重要的大分子相互作用 组装和激活酶-单链DNA复合体所需的，以及 用于用其他蛋白质取代单链DNA中的HDPs。在一个更多的 一般意义上，确定所需的大分子相互作用 单链DNA上一种蛋白质被另一种蛋白质取代应该有助于 我们对发生的许多其他蛋白质置换反应的理解 在各种遗传过程中，包括移位 转录因子引起的核小体与一种蛋白质的置换 在信使的处理和细胞内运输过程中被另一个人 RNA。

The broad goal of this research program is to elucidate the molecular mechanisms by which enzyme complexes on single-stranded DNA are assembled and activated. The interaction of specific enzymes with ssDNA is an essential feature of the mechanisms of DNA replication. recombination, and repair that occur in all living cells. The assembly and activation of enzyme-ssDNA complexes is complicated, however, by the presence of helix- destabilizing proteins (HDPs) that bind tightly and cooperatively to ssDNA. To bind to ssDNA and activate its catalytic activities, an enzyme must first displace HDPs, an energetically unfavorable process. Therefore special HDP displacement mechanisms appear to be required to load an enzyme onto a preexisting HDP-ssDNA matrix. We propose to study these displacement and loading mechanisms by investigating the molecular steps involved in the assembly and activation of two different model enzyme- ssDNA complexes: that of a DNA helicase, the bacteriophage T4 gene 41 protein (gp41), and that of a general recombinase, the T4 uvsX protein. The assembly and activation of each enzyme-ssDNA complex, appears to require the protein-ssDNA and/or protein-protein interactions of as many as three different phage proteins: the enzyme itself, an enzyme-specific assembly factor, and a specific HDP-- the T4 gene 32 protein (gp32). Our specific objective is therefore to dissect the protein-ssDNA and protein- protein interactions involved in the loading of gp41 and uvsX onto gp32- ssDNA. This will be accomplished by analyzing the interactions of the two assembly factors with ssDNA, with gp32 and with their enzyme partners. Protein-ssDNA interactions will be examined directly by fluorescence spectroscopy and indirectly by coupled spectrophotometric assay. Protein- protein interactions will be examined by a combination of protein affinity chromatography and sedimentation methods and by the isolation and characterization of specifically altered proteins. It is expected that these studies will identify the important macromolecular interactions required for the assembly and activation of enzyme-ssDNA complexes, and for the displacement of HDPs from ssDNA by other proteins. In a more general sense, identifying the macromolecular interactions required for the displacement of one protein by another on ssDNA should contribute to our understanding of many other protein displacement reactions that occur during the various genetic processes, including the displacement of nucleosomes by transcription factors and the displacement of one protein by another during the processing and intracellular transport of messenger RNAs.