Structural Studies of the SV40 Large T Antigen

SV40 大 T 抗原的结构研究

• 批准号: 6511637
• 负责人: MARGARET S VANLOOCK
• 金额: $ 3.83万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6511637
• 关键词: DNA; DNA replication; DNA replication origin; computer simulation; conformation; cryoelectron microscopy; enzyme mechanism; enzyme model; enzyme substrate; helicase; image processing; intermolecular interaction; model design /development; molecular dynamics; molecular site; protein structure function; simian virus 40; site directed mutagenesis; structural biology; transmission electron microscopy; tumor antigens; virus protein; virus replication

The Simian Virus 40 (SV40) has served as a powerful tool in understanding the detailed events involved in the initiation and regulation of DNA replication. Interesting, only one protein, Large T antigen (T-ag), is necessary for the initiation viral replication. The SV40 T-ag is a multifunctional protein that serves to force the host cell into S- phase, initiate viral replication, and regulate its own expression at the level of transcription. This protein is known to bind to both the SV40 core origin DNA and to synthetic DNA replication forks. Studies in Dr. Nick Cozzarelli's group have shown that in the presence of synthetic replication forks and ADP, T-ag forms two distinct complexes with differing gel mobilities. One primary goal the proposed research will be to generate 3-D reconstructions of these complexes using both negative stain and cryo-EM techniques. The resulting reconstructions will aid In understanding the DNA binding and helicase activities of Large T-ag. Large T-ag is composed of several interrelated functional and structural domains. Amino acid sequence maps of these domains have been defined; however, the location of these domains in the 3-dimensional structure of the Large T-ag is unknown. The second primary goal of the proposed research is to generate numerous 3-D reconstructions of mutant and truncated forms of the SV40 T-ag and of T-ag complexed with a variety of cellular proteins. These reconstructions will be used to develop a detailed 3-D map and structural models for the domain organization of T-ag that will help identify interactions that are necessary for Large T-ag function.

猿猴病毒40（SV 40）已成为了解DNA复制启动和调控的详细事件的有力工具。有趣的是，只有一种蛋白质，大T抗原（T-ag），是启动病毒复制所必需的。SV 40 T-ag是一种多功能蛋白，其用于迫使宿主细胞进入S期，启动病毒复制，并在转录水平上调节其自身的表达。已知该蛋白质结合SV 40核心起始DNA和合成DNA复制叉。Nick Cozzarelli博士小组的研究表明，在合成复制叉和ADP的存在下，T-ag形成了两种具有不同凝胶迁移率的不同复合物。一个主要的目标，拟议的研究将是产生三维重建这些复合物使用负染色和冷冻EM技术。由此产生的重建将有助于理解大T-ag的DNA结合和解旋酶活性。大T-ag由几个相互关联的功能和结构域组成。这些结构域的氨基酸序列图已被定义;然而，这些结构域在大T-ag的三维结构中的位置是未知的。拟议研究的第二个主要目标是生成SV 40 T-ag和与各种细胞蛋白复合的T-ag的突变体和截短形式的许多3-D重建。这些重建将用于开发T-ag结构域组织的详细三维地图和结构模型，这将有助于识别大T-ag功能所需的相互作用。